Metabolism: Growing
­ ustaining
Membranes, S
Cells
Chapter
9
“Organisms are not mere assemblages of genes, whether inherited vertically
or laterally, but cells in which there is a mutualistic cooperation of genomes, membranes, skeletons and catalysts that together make a physically and
functionally coherent unit capable of reproduction and evolution.”
(Thomas Cavalier-Smith, Nature 446:257, 2007)
T
his chapter discusses the metabolic roles cell membranes play in biosynthesis and energy metabolism in cells, the functional units of life.
Membranes integrate the activities of the various metabolic compartments
of a cell, are central players in extracting energy from the environment, and
direct their self-renewal through synthesis of lipids using their own membrane-bound enzymatic machinery. Across generations, membranes maintain their own lineage through contiguous growth and division, both at the
level of the cell and individual organelles.
Compartmentation of Metabolism
Membranes are an integral part of cellular metabolism
To say that the cell is the functional unit of an organism can best be understood on the basis that membranes form self-enclosed barriers that allow
cells to maintain distinct metabolic and genetic identities. By forming
boundaries around cellular compartments such as the cytosol and the
lumen of various organelles, membranes help partition cellular metabolism
into functional units (Figure 9.1). For instance, as shown in Figure 9.1, the
branching pathways from Ain → B and Ain → D in the cytosol are physically
separated from the circular pathway C in the lumen of the mitochondrion.
Compartmentalization does not mean that pathways are isolated. Rather,
the cell connects pathways using membrane transporters (for example,
those that mediate Aout → Ain and B → C) that shuttle metabolic intermediates between aqueous compartments. And by controlling the transporters, membranes control the metabolic flow between pathways—that is, how
quickly and efficiently a particular pathway, or set of pathways, processes a
given amount of metabolites and energy.
Membranes, however, are more than spatial organizers of metabolic
compartments. They maintain entire pathways within their own twodimensional matrix, the same way cells organize transporters, receptors,
and cell adhesion molecules into specialized membrane domains that
variably form cell junctions and domains for exocytosis and endocytosis, as well as tubular membrane extensions like microvilli for nutrient
absorption, secondary cilia and flagella for movement, and primary cilia,
cytonemes (filapodia), axons, and dendrites for cell signaling. In other
words, many metabolic reactions operate within or along the surface of
cell membranes as shown for pathway R → S in Figure 9.1. Well-studied
CellMembranes ch09.indd 325
Aout
OUTSIDE
CYTOPLASM
D
Ain
MEMBRANE
R
LUMEN
S
C
B
Figure 9.1 Membranes and metabolic
pathway organization in eukaryotic cells.
Membranes are integral parts of cellular
metabolism, separating and connecting
aqueous pathways that operate in separate
compartments. The branched pathways
A → B and A → D are separated from
circular pathway C, but can be connected
by importing the end product of pathway
A → B into the organellar subcompartment.
Pathways are connected by substratespecific transporters (active or passive) and
import nutrients into the cell. Membranes
have their own pathways (for example,
R → S) with metabolic functions that go
beyond mere transport.
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Chapter 9: Metabolism: Growing Membranes, ­Sustaining Cells
membrane-bound pathways include electron-transport chains and ATP
synthases in cellular respiration and photosynthesis, the biosynthesis
and degradation of membrane lipids and proteins, as well as membrane
trafficking and signaling pathways. Membrane-bound pathways require
membrane-bound catalysts. Accordingly, enzymes in a pathway of type
R → S include transmembrane and surface-bound proteins. They convert
membrane-bound substrates into membrane-bound products, but also
convert water-soluble substrates into membrane-bound products, and
vice versa, allowing soluble and membrane-bound pathways to interact
with each other.
Figure 9.2 Compartmentation of fatty
acid metabolism in liver. Fatty acid
metabolism is a primary metabolic activity
of liver cells. The liver is able to convert
sugars into fats, exporting the latter for use
by other cells or for long-term storage (as
low-density lipoprotein [LDL] particles). In
this process, the glycolytic end product
pyruvate is imported into the mitochondrial
matrix compartment where it is converted
to acetyl-CoA. Acetyl-CoA is exported
to the cytoplasm via the citrate shuttle,
a step that couples fatty acid synthesis,
an energy-demanding process, to the
Krebs cycle, a central energy-producing
pathway in the mitochondrial matrix
compartment. Thus, fatty acid synthesis
is coupled to energy availability. Fatty
acid synthesis is a cytoplasmic pathway.
The precursor for fatty acid synthesis
is acetyl-CoA, which is produced in the
mitochondrial matrix compartment from the
degradation of pyruvate, amino acids, or
fatty acids. Acetyl-CoA is combined with
oxaloacetate (OAA) to produce citrate.
Citrate is exported into the cytoplasm,
where it is split back into acetyl-CoA and
OAA. The latter is recycled back into the
mitochondrion, via malate, to pyruvate
metabolism, to convert cytosolic NADH
into NADPH, the energy source of fatty
acid synthase (FAS) enzymes. The end
product of FAS is the saturated 16-carbon
fatty acid, palmitic acid (C16:0). To prevent
the newly synthesized fatty acids from
being transported into the mitochondrion
for degradation (via beta oxidation),
the committed precursor of fatty acid
synthesis, malonyl-CoA, inhibits carnitinepalmitoyl transferase 1A (CPT1A) on the
outer membrane of the mitochondrion
(see Figure 9.3 for details). This prevents
newly synthesized palmitic acid (C16:0)
from import into the mitochondrial
compartment; it is instead processed by
elongase and desaturase enzymes of
the smooth endoplasmic reticulum first
to stearic acid (C18:0) and then to oleic
acid (C18:1). Palmitic, stearic, and oleic
acids are found in various combinations in
phospholipids and triacylglycerols (TAGs).
The latter can be bundled with cholesterol
and lipoproteins into LDL particles and
exported from the liver cell. G3P, glycerol
3-phosphate; PA, palmitic acid; DAG,
diacylglycerol.
CellMembranes ch09.indd 326
Membranes contribute to the spatial organization
of metabolic pathways
Cells have evolved efficient ways to functionally organize cellular metabolism into biosynthetic (anabolic) pathways consuming cellular energy and
‘separate’ them from degradative (catabolic) pathways that produce cellular energy. Functional separation occurs at the level of protein regulation,
where enzymes can be switched on and off as needed, and regulation of
gene expression, controlling the synthesis of enzymes for biosynthetic and
degradative pathways. Eukaryotic cells, by virtue of their multiple compartments, have also evolved spatial organization by operating biosynthetic
and degradative pathways in different membrane-bound compartments.
A well-studied example of pathway compartmentation is the physical
separation of fatty acid synthesis and degradation (beta oxidation) in liver
(Figure 9.2). The synthesis of palmitic acid, a saturated fatty acid with sixteen carbons (metabolic symbol C16:0), is carried out by the cytoplasmic
fatty acid synthase (FAS) protein complex. Further elongation and desaturation reactions, as well as incorporation of the fatty acids into phospholipids and triacylglycerols (fats and oils), are carried out by membrane-bound
enzymes of the smooth endoplasmic reticulum (ER). The smooth ER exports
triacylglycerols and cholesterol as low and very-low-density lipoprotein
(LDL, VLDL) particles via the secretory pathway for delivery to other cells in
the body. Degradation of fatty acids, such as the ones gained from the diet
(if not directly incorporated into membrane phospholipids or fats), occurs
G3P
lyso-PA
PA
glucose
C18:1
C18:0
C16:0
pyruvate
acetyl-CoA
+
OAA
BETA
OXIDATION
–
DAG
TAG+
cholesterol
LDL
acyl-CoA
FAS
citrate
pyruvate
malonyl-CoA
acetyl-CoA
pyruvate
citrate
malate
NADPH
OAA
NADH
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Compartmentation of Metabolism
327
in the matrix compartment of the mitochondrion (or in the peroxisome for
very-long-chain fatty acids of 22 carbons or longer) after transport across
the double-membrane system of the mitochondrion.
Fatty acid import into the mitochondrion is inhibited by malonyl-CoA, the
committed precursor during fatty acid synthesis. As fatty acid breakdown
and synthesis are completely separated into two different metabolic compartments—the mitochondrial matrix and the cytosol—the regulation of
a single protein, carnitine-palmitoyl transferase 1A (CPT1A), an enzyme
bound to the cytoplasmic leaflet of the outer mitochondrial membrane, is
sufficient to prevent a metabolic short circuit (Figure 9.3). Thus, liver cells
have a simple and efficient way to prevent degradation of newly synthesized
fatty acids without having to remove all the enzymes involved in beta oxidation, only to rebuild them later when metabolic needs change.
Pathway compartmentation is not and cannot be the only means of preventing wasteful concomitant activity of fatty acid synthesis and degradation.
Genetic control of protein levels for metabolic pathways allows coordination of lipid metabolism in the liver with supply and demand of the whole
organism. Genetic regulation is also essential for cellular differentiation in
multicellular organisms, explaining the metabolic and structural differences
between fat, liver, and muscle cells in our body. Last, but not least, genetic
(A)
CoA
(B)
acyl-CoA
carnitine
CPT1A
CYTOPLASM
acylcarnitine
VDAC
acyl-carnitine
INTERMEMBRANE
SPACE
ge
r
carnitine
OUTER
MEMBRANE
exc
ha
n
INNER
MEMBRANE
acylcarnitine
CPT2
CoA
carnitine acyl-CoA
MATRIX
carnitineacyl-carnitine
exchanger
homology model
bovine
ADP/ATP
carrier with lipid
and inhibitor
Figure 9.3 Fatty acid import into the mitochondrial matrix. (A) The import
of activated fatty acids (carried by carnitine as acyl-carnitine) is catalyzed by
the carnitine-acyl-carnitine exchanger (SLC25A20), a transporter of the inner
mitochondrial membrane. This transporter requires that the fatty acids are first
transferred from their CoA carrier to the amino acid carnitine as a substrate carrier.
This transfer is catalyzed by the carnitine-palmitoyl transferase (EC 2.3.1.21; ‘liver’
CPT1A, ‘muscle’ CPT1B, and ‘brain’ CPT1C) located on the cytosolic side of the
outer membrane. Malonyl-CoA inhibition works by blocking acyl-carnitine formation.
Acyl-carnitine is transported across the outer membrane through voltage-dependent
anion channels (VDAC; mitochondrial porin; PDB accession 2JK4) to reach the
inner-membrane carnitine-acyl-carnitine exchanger. This carrier is an antiporter
exchanging mitochondrial carnitine for a cytoplasmic acyl-carnitine. Carnitinepalmitoyl transferase CPT2 (PDB accession 2H4T), a monotopic membrane protein
associated with the matrix surface of the inner membrane, moves the fatty acid from
the carnitine to the CoA carrier, completing substrate import. (B) The structure of the
carnitine-acyl-carnitine exchanger (PDB accession 2BMN) has been modeled after
the conserved domain (pfam00153) it shares with mitochondrial carrier proteins as
exemplified by the ADP/ATP antiporter (PDB accession 2C3E).
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Chapter 9: Metabolism: Growing Membranes, ­Sustaining Cells
regulation is of great importance in the single compartments of prokaryotes.
Not able to exploit spatial separation, bacteria depend on genetic up- and
down-­regulation of the fatty acid degradation and biosynthesis pathways to
prevent a metabolic short circuit (Figure 9.4). Bacteria express the fatty acid
degradation regulatory element FadR, a transcription factor that controls the
expression of the genes for enzymes in fatty acid metabolism. FadR inhibits
the expression of enzymes in the fatty acid degradation pathway (beta-oxidation genes), but activates fatty acid synthase genes (fabA and fabB). The
presence of long-chain fatty acids reverses this regulation. When fatty acylCoA metabolites are plentiful as they are taken up from the growth medium,
they bind to FadR, which is then unable to bind to the regulatory elements
(promoters) of the corresponding genes. In a negative-feedback loop, fatty
acids that are no longer used for membrane and triacylglycerol synthesis
accumulate in the cytoplasm and trigger the synthesis of proteins for their
degradation and, at the same time, suppress the expression of fatty acid synthase genes.
(A)
beta oxidation
–
fatty acid synthase
fatty acid
acyl-CoA
fatty acid
acyl-CoA
+
acetyl-CoA
and energy
FadR
(B)
The transport metabolon: when metabolic pathways span
across compartments
PROMOTING BIOSYNTHESIS
FadR
fad
FadR
fabA
fabB
β-ox genes
degradation
OFF
FAS II genes
synthesis
ON
PROMOTING DEGRADATION
acyl-CoA
fad
fabA
fabB
β-ox genes
degradation
FadR+acyl-CoA
FAS II genes
synthesis
ON
OFF
Figure 9.4 Genetic regulation of fatty
acid metabolism in bacterial single
compartments. (A) Fatty acid synthesis
and degradation in bacteria occur in
the same cytoplasmic compartment
and are regulated by genetic control via
the transcription factor FadR. (B) The
transcription factor FadR suppresses genes
needed for fatty acid degradation (betaoxidation [β-ox] genes) and stimulates
expression and synthesis of two key
enzymes in fatty acid synthesis, FabB and
FabA. These enzymes are responsible for
fatty acid desaturation and elongation.
FadR is recognized by long-chain fatty acylCoA molecules and, as a result of binding
to them, can no longer bind to the promoter
regions. As a result, fabA and fabB genes
are repressed while beta-oxidation genes
are activated. Thus, excess fatty acids from
synthesis or uptake from the surroundings,
if no longer needed for phospholipid and
triacylglycerol synthesis, trigger their own
degradation.
CellMembranes ch09.indd 328
Not all pathways are neatly organized within one individual compartment
and connected by transporters to other pathways. Instead, some pathways
operate across two or more compartments. In other words, some pathways
are segmented into pieces operating in different compartments in eukaryotic
cells, where membrane transporters become part of the pathway itself, as is
well described for gluconeogenesis in liver (Figure 9.5A). Gluconeogenesis
produces free glucose from amino acids and its main function is to
replenish blood glucose levels in the absence of dietary carbohydrates.
Gluconeogenesis reverses glycolysis, the pathway that breaks down glucose
to pyruvate in the cytoplasm of the cell. Glycolysis is a linear pathway that
includes the sequential operation of ten enzymes, all of which are located
in the cytoplasmic compartment. During gluconeogenesis, seven of these
glycolytic enzymes operate in reverse, but three key regulatory enzymes—
hexokinase, phosphofructokinase, and pyruvate kinase—are replaced by a
set of other enzymes, some located in the mitochondrial matrix compartment and the ER membrane.
Gluconeogenesis starts with pyruvate, which can be obtained from the
amino acid alanine. Instead of being converted to phosphoenolpyruvate by
pyruvate kinase, pyruvate is transported into the mitochondrial matrix compartment, first across the outer membrane through VDAC pores and then
the inner membrane by the mitochondrial pyruvate carrier (MPC1 and 2).
Like the carnitine-acyl-carnitine translocase (SLC25A20 gene), which is
involved in fatty acid import, the pyruvate carrier is just one of many innermembrane transporters that couple the energy-producing pathways in the
mitochondrial matrix with the cytoplasmic compartment (Table 9.1).
Once in the matrix compartment, pyruvate is converted to oxaloacetate by
pyruvate carboxylase (Figure 9.5B). Oxaloacetate is then reduced to malate
to capture mitochondrial energy from NADH. Malate is subsequently
exported (as there is no oxaloacetate transporter) by the dicarboxylate carrier (SLC25A10), a malate–phosphate antiporter, into the cytoplasm. There,
malate is oxidized back to oxaloacetate, freeing-up the captured reducing
power (energy) in the form of cytoplasmic NADH. NADH is the energy-rich
co-substrate of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the
key enzyme in glycolysis/gluconeogenesis. The overall effect of the mitochondrial bypass reaction is to export high-energy molecules for the reductive biosynthesis of glucose from pyruvate. At times of carbohydrate shortage,
these high-energy molecules (NADH) are plentiful in mitochondria—for
22/04/15 1:30 PM
Compartmentation of Metabolism
example, from beta oxidation and the citric acid cycle (or Krebs cycle)—but
not in the cytoplasm.
Finally, the two kinase steps—hexokinase and phosphofructokinase—are
reversed by corresponding phosphatases. Fructose 1,6-bisphosphatase is a
cytoplasmic enzyme, but glucose 6-phosphatase activity is located within
the ER lumen to separate it from hexokinase activity, thus preventing a
short circuit that potentially wastes ATP (Figure 9.5C). This compartmentation requires transport of glucose 6-phosphate (G6P) into the ER lumen
by the G6P/Pi antiporter (G6PT1; solute carrier family SLC37A4). The luminal phosphatase activity releases the free phosphate needed to import
more G6P from the cytoplasm. Free glucose is then released back into the
cytoplasm through a still-to-be-identified transporter and diffuses across
the plasma membrane into the extracellular fluid. Candidates for the ER
transporter include the GLUT8 uniporter (SLC2A12) and the sugar efflux
transporter SWEET1 (SLC50A1). Both transport proteins are expressed in
ER and Golgi membranes, where they facilitate import of monosaccharide
precursors into the organelle lumen for lipid and protein glycosylation
reactions.
glucose
(A)
glucose
ER
glucose 6-phosphate
fructose 1, 6-bisphosphate
PEP
pyruvate
mitochondrion
(B)
oxaloacetate
malate
NADH/H+
NAD+
PEP + CO2
oxaloacetate
malate
needed
for
GAPDH
NADH/H+
NAD+
(C)
G6P
mitochondrion
Figure 9.5 Compartmentation of glycolysis/gluconeogenesis in liver
cells. (A) The synthesis of glucose (gluconeogenesis) in liver cells depends
on three metabolic compartments: the cytoplasm, mitochondrial matrix, and
endoplasmic reticulum (ER) lumen. Three key kinases in glycolysis—hexokinase,
phosphofructokinase, and pyruvate kinase—catalyze nonreversible reactions and
are replaced by other enzymes in gluconeogenesis. The phosphofructokinasecatalyzed reaction is reversed by fructose bisphosphatase, another cytoplasmic
enzyme. The reactions catalyzed by pyruvate kinase and hexokinase, however, are
reversed using enzymes located in the mitochondrial matrix and luminal side of the
ER membrane. (B) Pyruvate conversion to phosphoenolpyruvate (PEP) requires
a detour into the mitochondrial matrix. Conversion of mitochondrial pyruvate to
oxaloacetate and malate captures energy from the reducing power of NADH
(obtained from the Krebs cycle and beta-oxidation reactions burning fats). After
export into the cytoplasm, malate is oxidized back to oxaloacetate, releasing the
captured energy as cytoplasmic NADH, the energy co-substrate needed for the
reversal of the cytoplasmic glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
reaction. Oxaloacetate is decarboxylated to PEP, completing the reversal reaction of
pyruvate kinase. (C) Dephosphorylation of glucose 6-phosphate (G6P) is carried out
on the luminal side of the ER membrane. G6P is transported into the ER lumen by
the G6P/Pi antiporter (G6PT1), member 4 of the solute carrier family 37 (SLC37A4).
There, glucose 6-phosphatase (G6PC) removes the phosphate and produces free
glucose. The release mechanism of glucose from the ER is not fully understood and
is possibly facilitated by either a glucose uniporter (GLUT8) or SWEET1, an analog
of plant and microorganism sugar transporters.
329
Pi
CYTOPLASM
SL C3 7A4
G6PC
?
ER LUMEN
Pi
G6P
glucose
The close association of transporters with metabolic enzymes is often found
to form dynamic protein complexes called transport metabolons. Transport
metabolons shorten the diffusion distance for substrates from transporter to
enzyme, greatly enhancing the efficiency and speed of the pathway reaction.
Often, they are used to rapidly capture a metabolite as it enters the cell. An
example discussed earlier is the phosphotransferase complex in bacterial
group translocators for sugar intake (see Figures 7.6 and 7.27).
Bacteria also make use of compartmentation
While compartmentalization is an established feature of eukaryotic cells, it
is also found in some bacteria. Most notably, Gram-negative bacteria have
a periplasmic space between the inner and outer membrane. This space
serves as a sorting chamber for absorption and secretion (see Chapter 7).
True intracellular compartments akin to eukaryotic membrane-bound
organelles are found only in a few prokaryotes, but happen to be a characteristic feature of planctomycetes bacteria, a group of freshwater and marine
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Chapter 9: Metabolism: Growing Membranes, ­Sustaining Cells
Table 9.1 Mitochondrial inner membrane carriers
Gene name
Other names
Function
Porter type
MPC1/2
Pyruvate carrier
Links glycolysis to the Krebs cycle, exchange for OH–
Antiporter
SLC25A1
Tricarboxylate transporter,
citrate carrier, CTP
Citrate plus a proton in exchange for
another tricarboxylate-H+, a dicarboxylate, or
phosphoenolpyruvate
Antiporter
SLC25A2,15
Ornithine transporters
Exchanges ornithine for arginine in urea cycle
Antiporter
SLC25A3
Phosphate carriers, PHC
Phosphate/H+
Phosphate/OH–
Symporter
Antiporter
SLC25A4,5,6,31
ADP/ATP translocase
Import ADP and export ATP energy into cytosol
Antiporter
SLC25A7,8,9,14,27
Uncoupling proteins, UCP
Depletes proton gradient
Antiporter
SLC25A10
Dicarboxylate transporter
Malate and succinate in exchange for phosphate,
sulfate, and thiosulfate
Antiporter
SLC25A11
Oxoglutarate carrier
Oxoglutarate/malate carrier
Antiporter
SLC25A12,13
Aspartate/glutamate carriers
Amino acid exchange
Antiporter
SLC25A16,42
Grave’s disease carrier (GDC) protein
Coenzyme A transporter
Antiporter
SLC25A18,22
Glutamate/H+ symporter
Amino acid transport
Symporter
SLC25A19
Thiamine pyrophosphate carrier
Transports thiamine pyrophosphate into matrix
Uniporter?
SLC25A20,29
Carnitine-acyl-carnitine translocase,
CACT
Imports fatty acids into matrix
Antiporter
SLC25A21
Oxodicarboxylate carriers
Transports oxoadipate, a common intermediate
in the catabolism of lysine, tryptophan, and
hydroxylysine
Uniporter?
SLC25A23,25
ATP/Pi carriers, ACP
Responsible for the net uptake or efflux of adenine
nucleotides into or from the mitochondria
Antiporter
SLC25A32
Folate transporter
SLC25A33,36
Pyrimidine nucleotide carrier
Uniporter
Nucleotide transporter
Uni- and
antiporter
SLC25A numbers not listed are genes/proteins with unspecified function but belonging to the mitochondrial transport family, and can be
peroxisomal transporters.
organisms. Members of this bacterial group contain a membrane-bound
nuclear body consisting of two membranes—the cytoplasmic membrane
and the intracytoplasmic membrane surrounding the riboplasm and containing the fibrillar DNA-containing nucleoid, an intracellular structure
reminiscent of a nucleus. A subset of planctomycetes also has an ‘organelle’
called the anammoxosome (Figure 9.6). The anammoxosome is an energyproducing organelle. It synthesizes ATP using the energy from the oxidation
of ammonia and nitrates to molecular nitrogen (N2). The purpose of compartmentalization in this case seems to be protection of the cell’s DNA. The
anammoxosome has specialized ladderane lipids (see Figure 4.15C). Their
polycyclic fatty acid tails decrease both the fluidity and permeability of the
anammoxosome membrane, which is thought to prevent diffusion of nitric
oxide radicals into the riboplasm, where they can be damaging to proteins,
lipids, and nucleic acids.
When counting membranes as metabolic compartments proper, the cell
envelope of the Gram-negative bacteria can be considered to be made of
four compartments: the inner membrane, the periplasmic space, the outer
membrane, and the extracellular ‘space’ that includes fibrous extensions of
the bacterial cell wall (Table 9.2). A proteome analysis of Escherichia coli
K-12 substrain W3110 showed a cell envelope consisting of 1179 known and
putative proteins, or 28% of all 4213 proteins found in the genome of this
bacterium.
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Compartmentation of Metabolism
cell wall
plasma membrane
anammoxosome
paryphoplasm
intracytoplasmic
membrane
riboplasm
nucleoid
NH4+
hao
N2
N2H4
NO
NO2–
hh
4e–
cyt
331
Figure 9.6 The anammoxosome of
Candidatus Brocadia anammoxidans. The
anammoxosome is a specialized energyproducing organelle in the bacterial phylum
planctomycetes. Planctomycetes contain
at least two membranes: a cell membrane
and an intracytoplasmic membrane. The
intracytoplasmic membrane partitions
the cytoplasm into the ribosome-free
paryphoplasm (metabolism) and the
riboplasm, a nucleus-like compartment with
the highly organized bacterial chromosome
(nucleoid region) and ribosomes.
Ammonium-oxidizing bacteria (Candidatus
Kuenenia and Anammoxoglobus spp.)
have an additional energy-producing
compartment within the riboplasm—the
anammoxosome—which oxidizes ammonia
(NH4+) and nitrite (NO2−) to N2 and water.
The reaction feeds an electron-transport
chain producing ATP at the expense of a
proton gradient across the anammoxosome
membrane.
nir
cyt
cyt
3e–
Q
1e–
ATP
bc 1
ADP+P
6H+
3H+
A breakdown by function shows that 93% of all defense-related proteins are
found in the cell wall, protection against toxins and phages being one of the
cell wall’s primary jobs. And not surprisingly, a large proportion of envelope proteins are involved in transport. For various metabolic categories
(for example, carbohydrate transport and metabolism), the data in Table
9.2 pools transporters and enzymes together, reflecting our understanding that metabolic pathways can only be fully appreciated when integrating membrane transporters (transport metabolon). In fact, we have seen
earlier (see Figures 7.6 and 7.27) that bacterial phosphotransferase systems
couple sugar transport with kinase activity, a striking example of integrating transport with metabolism. This type of integration is also reflected at
the chromosomal level. One of the best-studied gene expression systems,
the bacterial lactose operon, is a genetic ‘unit’ of three structural genes that
include the lactose permease, a transporter, and two lactose-catabolizing
enzymes, β-galactosidase and β-galactoside transacetylase.
As whole-genome and proteome analyses often include many unknown,
novel genes and proteins (that is, predicted from sequence features alone),
allocation of proteins to each envelope localization is based on various structural factors such as α-helical topology (transmembrane, inner membrane),
discriminators of outer-membrane and β-barrel topology (transmembrane,
outer membrane), predictors of signal peptides and subcellular localization
signals, and annotations in knowledge databases (that is, collecting data
from published results). Consistent with the common results of exploring
whole genomes, which often show a large percentage of predicted genes to
be of unknown function, of all E. coli proteins identified in this study, 716 or
17% are hypothetical proteins or of unknown function. Of those unknown
proteins, a whopping 57% are predicted to be inner-membrane proteins.
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Chapter 9: Metabolism: Growing Membranes, ­Sustaining Cells
Table 9.2 Escherichia coli cell envelope proteome
% of all proteins in category
Functional category (COG classification)
Defense mechanisms
CP (2981)
IM (857)
PE (245)
OM (77)
EC (53)
7
83
10
–*
–*
Inorganic ion transport and metabolism
8
69
17
7
–
Carbohydrate transport and metabolism
54
35
10
1
–
Amino acid transport and metabolism
50
40
10
–
–
Nucleotide transport and metabolism
82
14
4
–
–
Secondary metabolism, transport
80
11
9
–
–
Cell-envelope biogenesis, OM
29
30
15
25
1
Intracellular traffic, vesicular transport
42
35
10
7
6
Cell motility and secretion
43
34
11
6
6
Energy production and conversion
50
33
17
–
–
Signal-transduction mechanisms
52
37
11
–
–
Cell division and chromosome partitioning
56
44
–
–
–
Transcription
61
28
7
3
2
Post-translational modifications, chaperones
63
21
13
–
3
Lipid metabolism
68
30
–
3
–
Coenzyme metabolism
87
10
2
1
–
General function prediction only**
65
30
5
1
–
Function unknown**
30
57
10
1
1
* Of course, the lipids of the outer membrane and the lipopolysaccharide-based oligosaccharides provide a physical barrier against invaders.
** Poorly characterized; the general function relates to a functional class only. CP, cytoplasm; IM, inner membrane; PE, periplasm; OM, outer
membrane; EC, extracellular; in parentheses, the number of proteins in the category. (Adapted from Díaz-Mejía JJ, Babu M & Emili A [2009] FEMS
Microbiol Rev 33:66–97; and Tatusov RL, Galperin MY, Natale DA & Koonin EV [2000] Nucleic Acids Res 28:33–36; see also NCBI Genome Escherichia
coli, http://www.ncbi.nlm.nih.gov/genome/167?project_id=161931.)
In other words, this particular study suggests that roughly 20% of the E. coli
genome codes for inner-membrane proteins, and that we know the function
for only half of them.
Membranes host their own metabolic pathways
It is one thing to integrate metabolic pathways by way of substrate transport across membranes; it is quite another to maintain an entire pathway
within the confines of the lipid bilayer. Like soluble metabolic pathways,
membrane-bound pathways are highly organized. The top-level organization is the presence of separate membranes with unique functionality. In
eukaryotic cells, metabolic functions are divided up into the many organellar membranes, while prokaryotic plasma membranes are an all-inone solution with a single membrane carrying out all membrane-bound
functions. At the mid-level organization, membrane-bound pathways
are localized to restricted membrane areas (domains) within the same
membrane; for example, the lipid rafts in plasma membranes; the smooth
and rough areas of the ER membranes; the apical and basolateral membranes in polarized epithelial cells; the tubular extensions of the plasma
membrane that form sensory ‘organelles’ like primary cilia, developmental filopodia, and neuronal dendrites; or the electrically excitable axonal
membranes of neurons and inwardly folded T-tubules of striated muscle
cells. These areas form membrane domains that carry out specialized metabolic functions.
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Compartmentation of Metabolism
(A)
NAD+
NADH/H+
O2+
red.
ox.
reductase
cytochrome
ox.
red.
red.
desaturase
oxygenase
ox.
red.
or
H2O+
OH
ox.
(B)
HMG-CoA
mevalonate
(C)
farnesyl
geranyl
isopentenyl/
dimethyl allyl
squalene
cholesterol
GPCR
ligand
α
PIP2
DAG
+
β
PLC
γ
+
PKC
IP3
G-protein
Within these domains, the basic pathway organization puts membrane proteins into functional protein complexes such as electron-transport chains,
biosynthetic pathways in lipid synthesis, and local signal-transduction pathways (Figure 9.7). For instance, the smooth ER membrane contains short
electron-transport chains made of reductases, cytochromes (P450 and
cytochrome b5), desaturases, and oxygenases. These enzymes use reducing power from NAD(P)H in the presence of molecular oxygen to oxidize
lipids. They introduce cis-double bonds (desaturation) to produce unsaturated fatty acids, and add oxygen units (oxygenation) to form the hydroxyl
groups that are found in lipid-based hormones like steroids, eicosanoids,
and jasmonates.
333
Figure 9.7 Membrane-bound pathways.
Membranes contain their own pathways
for energy metabolism, biosynthesis,
and signaling. (A) Biosynthetic electrontransport chains composed of desaturases
and oxygenases (hydroxylases) convert
carbon single bonds into cis carbon
double bonds (desaturation) or introduce
oxygen units to form hydroxyls. These
pathways operate in the presence of
molecular oxygen, which is reduced to
water in the process. The energy for the
reaction comes from NADH. Depending
on the reaction mechanism (desaturation,
monooxygenation, dioxygenation), none,
one, or two oxygens are incorporated
into the saturated lipid substrate. (red.,
reduction; ox., oxidation.) (B) Cholesterol
synthesis is a membrane-bound
pathway. The water-soluble substrate
hydroxymethylglutarate (HMG) is converted
to a lipid-bound precursor (isopentenylPP), which is used to form longer geranyl,
farnesyl, and squalene prenols. Squalene
cyclization and oxygenation produces
cholesterol. (C) Some signal-transduction
pathways are also membrane-bound
pathways. A G-protein-coupled receptor
(GPCR)-activated G-protein stimulates
phospholipase C (PLC), which splits
phosphatidylinositol bisphosphate
(PIP2) into two signaling molecules: the
cytoplasmic inositol trisphosphate (IP3, a
second messenger) that activates calcium
release from the endoplasmic reticulum,
and membrane-bound diacylglycerol (DAG)
that activates protein kinase C (PKC). Both
G-protein and DAG signaling depend on
lateral diffusion along the cytoplasmic cellmembrane surface.
Another example of membrane-bound metabolism in the smooth ER is the
synthesis of prenols and cholesterol (Figure 9.7B). This pathway starts with
water-soluble substrates that are converted to membrane-bound products
through a series of reductase, kinase, synthase, and oxygenase reactions. The
committed step occurs on the cytoplasmic side of the ER membrane. The
enzyme HMG-CoA reductase (the target of cholesterol-lowering drugs like
Lipitor® and Crestor®) binds hydroxymethylglutarate (HMG) and reduces
it to mevalonate. Subsequent phosphorylation and decarboxylation steps
convert mevalonate to the membrane-soluble isopentenyl-PP, the universal five-carbon precursor for prenol and sterol biosynthesis. Elongation
produces multiple prenol intermediates like geranyl (10 carbons), farnesyl
(15 carbons), and squalene (30 carbons), which serve as protein anchors and
precursors for heme, dolichol, and quinone synthesis. Squalene is converted
to cholesterol through multiple oxygenation and cyclization reactions.
To put the membrane-bound organization of these metabolic enzymes into
a proper context, these pathways are topologically equivalent to some signal-transduction pathways that operate along the membrane surface (see
Box 8.1). Diffusible, lipid-anchored G-protein subunits activate enzymes
like kinases and phospholipases and membrane channels. For example
(see Figure 9.7C), active phospholipase C hydrolyzes phosphatidylinositol bisphosphate (PIP2) into its free head group, the water-soluble second
messenger molecule inositol trisphosphate (IP3), and membrane-bound
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Chapter 9: Metabolism: Growing Membranes, ­Sustaining Cells
diacylglycerol (DAG) (see also Figure 8.26). DAG itself is an activator of
another membrane-bound enzyme, protein kinase C. Both the enzymatic
and signaling pathways interact with diffusible substrates in the adjacent
aqueous compartments.
Membrane enzymes come in all forms and shapes
(and reaction mechanisms)
Membrane-bound catalysis is no less diverse than cytosolic metabolism.
These membrane reactions contribute to the synthesis of phospholipids,
sterols, steroid hormones, bile acids, waxes, quinones, hemes and chlorophylls, carotenoids, and eicosanoids and to the glycosylation of proteins and
lipids. Many membrane enzymes are hydrolases including proteases, lipases,
and glycosidases involved in the degradation, conversion, and activation of
peptide, oligosaccharide, and lipid substrates (Table 9.3). Other well-characterized enzymes include oxido-reductases and transferases involved in
electron-transport systems, lipid desaturation, and oxygenation. And many
biosynthetic pathways would not be complete without the lyases, isomerases, and ligases involved in rearrangements of molecular structures such as
elongation, cyclization, methylation, branching, and polymerization.
Obviously, membrane-bound enzymes have distinctive requirements, demand­
ing special membrane-anchoring domains (transmembrane, monotopic, lipidanchored) and active sites for the biosynthesis and degradation of hydrophobic
and hydrophilic substrates. While the hydrophobic ‘environment’ suggests the
need for novel catalytic mechanisms due to the location in a lipid bilayer, the
active sites of membrane-bound enzymes simply adapt to their environment by
creating an active site that is accessible to both polar and nonpolar substrates.
Many reactions catalyzed by membrane-bound enzymes occur at the
bilayer surface, such as those carried out by phospholipases and oxygenases. For hydrophobic substrates, these enzymes provide an access ‘tunnel’ between the membrane surface and a hydrophobic binding pocket
for catalysis. For lipases that target the polar head group of a lipid, such as
phospholipase C, a simple hydrophobic anchoring of a membrane-binding domain will position the active site near the lipid substrate. Hydrolytic
activity within the hydrophobic core of the membrane has been described
for transmembrane proteases, most notably presenilins that are thought
to be involved in Alzheimer’s disease through hydrolysis of amyloid-beta
precursor protein. Initially, biochemists argued that it is unexpected to
find a reaction depending on water here, because the membrane core is
thought to be water-free. Often, the proteins provide a mixed hydrophobic–hydrophilic pocket or pore allowing access for both membrane-bound
(for example, lipids, transmembrane protein segments) and water-soluble
substrates (for example, water, NADH, mevalonate, glycerol 3-phosphate)
to the active sites through openings analogous to those found in transporters and channels. These active sites often contain polar or charged
co-factors such as FAD, Fe-heme, and Fe-S complexes. One should think
of the active sites of these transmembrane enzymes as a catalytic pocket
or half-pore with only one opening, facilitating substrate movement into
and out of the site, just the way transporters facilitate the movement of
substrates across the hydrophobic core of the membrane. The high-resolution structural work with ion channels in the 1990s has in fact demonstrated that nonpolar residues play critical roles inside these ‘water-filled’
pores, either increasing transport rate by reducing binding energy (potassium channels) or functioning as gates (nicotinic acetylcholine receptor).
Thus, a nonpolar environment for catalysis involving water (hydrolysis or
dehydration reactions) is entirely compatible with membrane-embedded
enzyme catalysis.
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335
Table 9.3 Diversity of membrane enzymes
Enzyme class
EC 1
Oxido-reductases
EC 2
Transferases
EC 3
Hydrolases
EC 4
Lyases
EC 5
Isomerases
EC 6
Ligases
Reaction
class (sample
subclass)
Function (sample enzyme)
Membrane
Association
Dehydrogenases
Quinol:fumarate reductase (Complex II)
Inner mitochondria, plasma
membrane bacteria
Transmembrane
Oxygenases
Cyt P450 monooxygenase (CYP1A1)
Endoplasmic reticulum
Monotopic
Cyclooxygenase (prostaglandin
synthase)
Endoplasmic reticulum
Monotopic–
luminal
Desaturases
Stearoyl-CoA desaturase
Endoplasmic reticulum
Transmembrane
Reductase
NADH-cytochrome b5 reductase
Endoplasmic reticulum
Lipid-anchored
(myristoyl)
Disulfide bond
oxido-reductase
Disulfide bond oxido-reductase-B
Bacterial Gram-negative
Transmembrane
Glycosyl
transferases
Peptidoglycan-biosynthesis-glycosyl
transferase MurG
Bacterial Gram-negative
inner membrane
Monotopic–
cytoplasmic
Acyl transferases
Elongation of long-chain fatty acids
(ELOVL family member 5)
Endoplasmic reticulum
Transmembrane
Proteases
Signal-peptidases
Bacterial Gram-negative,
Endoplasmic reticulum
Transmembrane
Rhomboid
Plasma membrane
Transmembrane
Site-2 protease families
(metalloproteases)
Plasma membrane,
Endoplasmic reticulum
Transmembrane
Lipases
Phospholipases A2, C
Plasma membrane
Transmembrane,
monotopic
Phosphatases
Alkaline phosphatase (intestinal)
Plasma membrane
Lipid anchored
Sulfatases
Estrone sulfatase
Rough endoplasmic
reticulum
Transmembrane
(luminal)
GTPases
Transducine
Eukaryotic plasma
membrane
Lipid anchor–
cytoplasmic
Glycohydrolases
Sucrase-isomaltase
Intestinal brush border
Transmembrane
(extracellular)
β-Glycosidase
Archaea
Monotopic
(extracellular)
Phosphorusoxygen lyases
Adenylyl cyclase
Plasma membrane
Transmembrane
Carbon-sulfur
lyases
Leukotriene-C4 synthase
Endoplasmic reticulum
Transmembrane
(cytoplasmic)
Endoperoxide
isomerases
Prostaglandin E synthase type 2
Golgi
Monotopic–
cytoplasmic
Lanosterol synthase
Endoplasmic reticulum
Monotopic–
cytoplasmic
E3 ubiquitin-protein ligase Itchy
homolog
Plasma membrane
eukaryotes
Monotopic–
cytoplasmic
Protein ligase,
synthase
Data from Orientations of Proteins in Membranes database, http://opm.phar.umich.edu/superfamilies.php
Like their water-soluble counterparts, membrane-bound enzymes are
regulated and access of substrates to their active sites is tightly controlled.
Two types of gating mechanisms have been observed: controlling access
from the surface or from the hydrophobic core. The monotopic prostaglandin H2 (PGH2) synthase (EC 1.14.99.1; PDB accession 1CQE) binds
its substrate from the embedded monolayer (Figure 9.8A). A functional
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Chapter 9: Metabolism: Growing Membranes, ­Sustaining Cells
dimer, PGH2 synthase has two active sites that operate in series—a
cyclooxygenase and a heme-dependent peroxidase. It converts a free arachidonic acid into an eicosanoid with a cyclic midsection and a series of
oxygenations, making the molecule less hydrophobic. The first active site
binds the free arachidonic acid (released from a phospholipid by phospholipase A2) by allowing the arachidonic acid substrate to diffuse from
its monolayer site into the enzyme active site. In the structure shown
in Figure 9.8A, the fatty acid binding site is occupied by an inhibitor of
PGH2, flurbiprofen. This first reaction is the cyclooxygenase reaction that
forms PGG2, which is then reduced to PGH2 by the heme-dependent
peroxidase active site.
Several transmembrane proteases catalyze their reactions with substrates
within the hydrophobic core of the membrane. For example, in the archaeal
metalloprotease mjS2P (PDB accession 3B4R), an intramembrane gate
controls the access of a membrane-spanning α helix of the targeted protein to the active site (Figure 9.8B). This site-2 protease is a homodimer,
with each monomer having six membrane-spanning helices surrounding
a centrally located Zn ion about 14 Å deep into the membrane. To bind a
substrate helix, one subunit in the protease widens its contact between
helix 1 and 6 to expose the incoming helix to the Zn co-factor in the active
site. Before opening the active site to the membrane core, the active site
is accessible to water molecules from the cytoplasmic side of the membrane through a polar channel. Water is a co-substrate in the reaction and
one water molecule interacts with the Zn ion prior to binding of the substrate transmembrane helix. When the protease changes its conformation
to allow the substrate helix to move in, the water molecule is trapped and
will interact with the peptide bond to be hydrolyzed. Access of polar substrates from the aqueous phase is gated by a loop and/or cap domain. This
mechanism has also been described for the rhomboid family of metalloproteases (with a structure related to E. coli GlpG serine proteases; PDB
accession 2IC8).
(A) SURFACE CATALYSIS BY A
MONOTOPIC OXIDO-REDUCTASE
(B) INTRAMEMBRANE PROTEOLYSIS
OH
O CH
O
O
PGH2
3
OH
90°
PGG2
ER LUMEN
H2O
O2
+ –
–
C
CYTOPLASM
AA
COOH
CH3
++ +
N
Figure 9.8 Active-site access in membrane-bound enzymes. Membrane-bound enzymes catalyze reactions on the membrane
surface (prostaglandin H2 synthesis) or within the hydrophobic core of the bilayer (intramembrane proteolysis). (A) Prostaglandin H2
synthase is a monotopic protein with two active sites facing the lumen side of the endoplasmic reticulum (ER) membrane. Arachidonic
acid (AA) is released from its phospholipid by phospholipase A2 activity and binds to the first of two active sites, a cyclooxygenase.
This first site catalyzes both a cyclization and peroxide formation of AA to produce prostaglandin G2 (PGG2); this is followed by the
peroxidase activity in the second active site, producing prostaglandin H2 (PGH2). (B) Intramembrane proteases cleave peptide bonds
of transmembrane α helices. Shown here is the structure of the site-2 intramembrane metalloprotease of the methanogenic archaeon
Methanocaldococcus jannaschii (PDB accession 3B4R). The protease is thought to bind a transmembrane helix by opening sidewise
and channeling water as a co-substrate from the membrane surface.
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Powering Cells: Membranes as Batteries
Membranes can extract, convert, and store energy
Living organisms are ‘open systems.’ To stay alive, they continuously extract
energy from their environment, converting the energy from food or light into
work—that is, forms of energy useful to their cells. Membranes play a central
role in these extraction and conversion processes, a role that goes beyond
the mere absorption of nutrients. Forms of energy that cell membranes can
‘work with’ include chemical, electrical, mechanical, and light energy. Cells
use their membranes to store and use electrochemical energy akin to batteries. To charge their ‘batteries,’ cells separate charges across their membranes
using energy extracted from molecules or light. Separating charges means
moving ions and building ion gradients. Ion gradients contain energy called
the electrochemical potential (see Box 6.1), which can be used to do work
(Figure 9.9). Types of work linked to ion gradients include membrane transport, biosynthesis of ATP, cell motility, and information processing through
electrical signaling.
The two most common ions linking electrochemical energy to cellular work
are the protons (hydrogen ions) and sodium ions. Protons and sodium ions
are often involved in the co-transport and absorption of nutrients into cells.
In plant cells, proton gradients help absorb sucrose into cells through a
sucrose permease (CscB) that couples sucrose transport to proton flow. CscB
is an oligosaccharide–H+ symporter and structural homolog of the bacterial
symporter lactose permease (lacY) from Escherichia coli. Ion exchangers like
Na+/H+ and K+/H+ transporters are crucial for pH regulation, volume control
(osmosis), and the electroneutral absorption and secretion of electrolytes.
Na+ and K+ ions are used for electrical signaling, including the propagation
of action potentials in neurons and muscle cells. Finally, proton gradients
are involved in prokaryotic cell motility, where proton (and in some cases
sodium) flow powers the rotor of the flagellar basal body of free-swimming
Na+
TRANSPORT
H+ or
Na+
nutrient
H+
SIGNALING
SYNTHESIS
H+ or Na+
Na+
+++
–––
+++
–––
+++
–––
ION
GRADIENT
K+
ATP
MOTILITY
H+
CellMembranes ch09.indd 337
H+ or Na+
ADP + Pi
Figure 9.9 Ion gradients as an energy
source for cellular processes. Cell
membranes have all the properties of an
electronic circuit found in battery-driven
devices used to power a lamp or machine.
They use their charged lipid bilayers
as batteries, powering ATP synthesis,
bacterial flagellar rotation, and transport
of many molecules that cells use as food.
Membranes use the electrochemical
potential—the free energy of an ion
gradient—to do work. Work in cells includes
activities such as membrane transport,
biosynthesis, cell motility, and electrical
signaling (information processing).
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Chapter 9: Metabolism: Growing Membranes, ­Sustaining Cells
prokaryotes. Because of its importance, the proton gradient is often referred
to as the ‘proton motive force’ or simply pmf.
(A)
INSIDE
OUTSIDE
(B)
Electron-transport chains in respiration and
photosynthesis are proton pumps
∂
α
β
α
ATP
b2
F1
ADP + Pi
INSIDE
H+ γ
ε
lipid
bilayer
FO
OUTSIDE
cn = ?
ab2
stator
rotor
H+
Figure 9.10 The ATP synthase is an
H+-driven rotor protein. (A) The structural
composition of an F1FO ATP synthase.
The F1 enzymatic part comes from bovine
mitochondria (PDB accession 1BMF). The
membrane-embedded FO-ATPase rotor
part is a C15 ring rotor from the flagellated
cyanobacterium Arthrospira platensis
(PDB accession 2WIE). (B) Schematic
drawing of the subunit composition of the
E. coli F1FO-ATPase with stator (ab2), rotor
(c-ring; the various numbers of subunits are
denoted as n), shaft (γ ε δ), and hexameric
enzyme complex (αβ). (Adapted from Jiang
W, Hermolin J & Fillingame RH [2001] Proc
Natl Acad Sci USA 98:4966–4971. With
permission from the National Academy of
Sciences.)
CellMembranes ch09.indd 338
The proton gradient is also used to synthesize ATP through an F-type ATP
synthase (structurally related to the proton-pumping V-type ATPases; see
Figures 7.24 and 7.25). This proton (and in some instances sodium)-driven
rotor complex is found in all domains of life: bacterial plasma membranes,
the inner membranes of mitochondria, and the photosynthetic membranes
of chloroplasts. An analogous A-type ATP synthase is found in archaea. The
F1FO-ATPases (and the related archaeal types) use a membrane-bound protein
ring structure (the FO portion) linked to a stator and a central shaft to mechanically alter the conformation of the enzyme domain (F1) (Figure 9.10). The
rotation of the membrane domain is driven by the sequential protonation–
deprotonation of a c-ring/stator (ab2δ) contact site. In the protonated state,
the contact site destabilizes and the ring ‘slips’ by one c subunit, exposing the
protonation site to a cytoplasmic/matrix exit, while opening a new protonbinding site on the extracellular/intermembrane-space side of the bilayer. The
stop-and-go movement of the c-ring driven by proton flow rotates the central gamma subunit (the shaft; γε), which induces a conformational change
in the F1 subunits. This mechanical distortion is an energy relay mechanism
between the membrane rotor and the enzyme catalytic site. The mechanical
energy of rotation is converted into chemical energy—that is, ATP.
Our main concern here is not how ion gradients are used to do cellular
work. These topics are discussed in Chapters 7 and 8. Instead, we want to
explore how proton gradients are built by one of the most powerful proton
pumps ever to evolve: the electron-transport chain. Proton pumps come
in various forms tapping into different energy sources: the light-driven
bacteriorhodopsins, the P- and V-type ATPases, the secondary active
transporters like Na+/H+ and K+/H+ exchangers, and the redox-powered
electron-transport chains. Bacteriorhodopsins are found in ‘purple membranes’ of photosynthetic archaea (not to be confused with purple bacteria
that use a chlorophyll-based electron-transport chain). While bacteriorhodopsins couple proton pumping directly to light energy, ATPases couple
proton pumping to chemical energy released from ATP hydrolysis. P-type
ATPases employ a simple catch-and-release mechanism, while V-type
ATPases use a rotary device. The latter, too, is built on a catch-and-release
mechanism, but instead of a single conformational switch, a rotating protein ring composed of nine to thirteen subunits releases the captured protons, one at a time, one on each subunit, upon interaction with the stator
unit, as in a musical-chair rotation (similar to the F1FO-ATP synthase that
operates in reverse). Finally, ion exchangers rely on the electrochemical
energy of Na+ and K+ gradients to transport H+ in an antiport carrier mechanism across the membrane.
Since ATP-driven pumps depend on the availability of ATP (and the secondary active transporters indirectly on the Na+/K+ pump or ATPase), the
question is what energy source other than ATP an organism might be able
to use. The answer is the redox potential of a multitude of small molecules
that serve as electron donors and acceptors and couple electron transport
to a proton flow across a membrane (Box 9.1). These electron-transport
chains create a series of steps between an electron donor (such as NADH
and FADH2) and a final electron acceptor (such as oxygen) (Figure 9.11A).
The reaction pathway for the electron flow uses a series of binding sites that
accept an electron/proton pair (e–/H+; reducing equivalent) or one electron
only. Alternating between e–/H+ and e–-only binding sites allows the capture
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339
and release of protons. When these alternate binding sites occur on opposite
sides of the membrane, protons are picked up by an electron via an e–/H+
binding site on side of the membrane and then released on the opposite side
of the membrane at an electron-only binding site. Thus proton pumping is
achieved. The protons move back through the ATP synthase and eventually
rejoin an electron at the terminal electron ‘sink’—for example, oxygen in the
respiratory electron-transport chain, which is reduced to water.
H+
H+
H+
H+
H+ H+
H+
e–
NADH and
FADH2
(B)
H2O O2
1
H+ –O
H+
2 2
H 2O
H+
H+
ATP ADP+Pi
H+ + H+ +
H
H
H+
e–
light
e–
H+
NADPH
H+
ATP
ADP+Pi
useful for work
H–C–H + O2 + NAD(P)H/H+
H–C–OH
RESPIRATION
(C)
PHOTOSYNTHESIS
Photosynthesis and respiration are among the most efficient and high-yielding energy-producing processes in modern organisms. Respiration converts
the high redox potential of C-H bonds in NADH/H+ and FADH2 into a proton gradient that drives ATP synthesis and discards the used electrons and
protons as O-H bonds in water (H2O) (Figure 9.11C). Photosynthetic membranes ‘reverse’ this process, investing light energy to raise the low potential
energy of electrons from O-H bonds in water into their high redox potential
in C-H bonds such as NADPH/H+ and ultimately sugars, fats, and amino
acids. The involvement of water in either process is crucial to explain the
large amount of energy that can be extracted or is invested. In this hydrogen economy, the C-H bond found in fats, carbohydrates, and proteins has
a high chemical potential (free energy) and is a form of energy useful to do
work, while the OH bond is a low-energy configuration and not useful to do
work. Respiration exploits the high-energy C-H bonds of carbon backbones;
photosynthesis reactivates the low-energy O-H bonds in water back into an
energy-rich carbon backbone. Simply put, the chemical energy (reducing
power of caloric nutrients) is where the CH bonds are. The presence of two
C-H bonds per carbon in fats and oils (H-C-H), as compared to one in carbohydrates (HO-C-H) and proteins, explains the higher caloric content of
lipids. It also explains why water—although involved in many biochemical
reactions—is, from an energy metabolism point of view, calorie-free.
(A)
free energy of e–/H+
In order to move through the electron-transport chains, electrons need to
be in a high-energy or activated state. While the electron-transport chain in
respiration taps into high-energy electrons extracted from high-energy molecules (NADH/H+, FADH2), photosynthesis first requires the input of light
energy to activate electrons found in water, or some other compounds such
as succinate, malate, H2, H2S, or S (Figure 9.11B). In oxygen-producing photosynthesis found in modern plants, electrons are extracted from water by a
manganese complex known as the oxygen-evolving center. The electrons are
then quickly shuffled to a magnesium ion in a chlorophyll molecule, where
the energy of a single photon activates a single electron, which then moves
along an electron-transport chain pumping protons. The proton gradient is
used to synthesize ATP by an F1FO-ATP synthase. At this point, the electron
needs a second round of light activation, which enables the final steps in the
electron-transport chain to reduce NADP+ to NADPH/H+.
H–O–H + CO2 + NAD(P)+
not useful for work
Figure 9.11 Nature’s hydrogen economy. (A) The electron-transport chain in respiration (prokaryotes, mitochondria) is a membranebound proton pump. It takes electrons and protons from NADH and FADH2 donors and uses the flow of electrons through a series
of enzyme complexes to move protons across the membrane. The complexes achieve this in part through their ability to alternate
electron flow between e–-only and e–/H+ binding sites. The electrons and protons are rejoined at the final electron acceptor, oxygen,
to remove the electrons in the form of H2O. The electron-transport chain allows proton accumulation on one side of the membrane,
producing a proton gradient that is exploited by an associate F-type ATP synthase to make ATP (a process dubbed chemiosmosis by
its discoverer, the biochemist Peter Mitchell, in 1961). (B) Photosynthesis in plants uses an electron-transport chain to produce ATP via
a proton gradient and captures high-energy electrons in the form of NADPH. Here, the electron is extracted from a low-energy state
(H2O) and is activated to a high-energy state using photon absorption in the special-pair chlorophylls (diamond). An electron-transport
chain pumps protons that are used for ATP synthesis. The electrons are rejoined with protons and removed from the electron-transport
chain by NADP+ reduction to NADPH. (C) Respiration and photosynthesis are key partners in life’s hydrogen economy, shuffling
electrons between low- and high-energy configurations (O-H and C-H bonds, respectively). Respiration is powered by high-energy
electrons from C-H bonds (from fats, sugars, and amino acids via NADH and FADH2), while photosynthesis uses light energy to
activate the low-energy electrons found in O-H bonds in water into the high-energy C-H configuration of sugars, fats, and amino acids
via NADPH. Note that in these biochemical processes, C-H bonds (prominent in fats) are high-energy configurations and O-H bonds
are low-energy configurations. This explains why fats with two hydrogens linked to a carbon (hydrocarbon, H-C-H) have double the
free energy (caloric content) of carbohydrates with only one hydrogen directly linked to a carbon (H-C-OH).
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Chapter 9: Metabolism: Growing Membranes, ­Sustaining Cells
BOX 9.1 Electron-transport chains as proton pumps
The respirasome is a supercomplex consisting of three
protein complexes: NADH dehydrogenase (complex I
or NADH:ubiquinone oxido-reductase), cytochrome
c reductase (complex III or cytochrome bc1 complex),
and cytochrome c oxidase (complex IV or ferrocytochrome c:oxygen oxido-reductase) (Figure 1). It is
a proton-pumping machinery that temporarily separates high-energy hydrogens extracted from NADH/
H+ into electrons and protons, where the downhill
electron transport supplies the energy for the uphill
transport of protons to produce a proton gradient. The
electron-transport chain is a linear reaction scheme
and complex IV eventually removes electrons, reducing molecular oxygen in the presence of protons into
water. An additional important player, succinate
dehydrogenase (complex II or succinate:FAD oxidoreductase; see Figure 9.12), is not included in the
respirasome, but contributes to the quinone pool by
feeding hydrogen directly from the Krebs cycle into
complex III.
(A)
H+ H+ H+ H+
cyt c
2H+
The three complexes of the respirasome are proton
pumps, with each complex employing its own unique
transport mechanism (shaded areas in Figure 1A). In
complex I, a carrier-type mechanism in the elongated
membrane-spanning domain is coupled to a conformational change (shown as ↔ in Figure 1) that is controlled
by electron flow from flavin mononucleotide via an iron–
sulfur (FeS) complex to the quinone binding site. These
antiporter-like domains have a piston-type catch-andrelease (or alternating access) mechanism controlled by
the reduction of the bound quinone to quinol.
The reduced quinol joins the quinone pool, which
helps shuttle protons from the matrix side of the inner
membrane to the intermembrane space (IMS). This is
achieved by positioning the oxidized quinone on the
matrix side of complex I, where it is reduced with two
electrons from NADH and two protons from the matrix
compartment. The reduced quinols dissociate and bind
to complex III at a high-affinity site at the IMS side of the
membrane.
H+ H+
H+
H+
e–
IMS
I
4H+
III
e–
Q
FeS
MATRIX
QH2 e–
heme
Q-pool
2H+
FMN
QH e
H+
H+
–
e–
CuA
e– heme
O
CH2 H C
3
H3C
IV
O
His
OH H3C
CuB-heme
O2
4H+ 2 H2O
(B)
4H+
OH
HO
N
N
O
Fe
N
N
HO
CH2
CH3
O
NADH/H+
(C)
complex I
complex III
complex IV
Figure 1 The mitochondrial respirasome as a proton pump. (A) The respirasome is a supercomplex consisting of three protonpumping protein complexes: NADH dehydrogenase (complex I or NADH:ubiquinone oxido-reductase), cytochrome c reductase
(complex III or cytochrome bc1 complex), and cytochrome c oxidase (complex IV or ferrocytochrome c:oxygen oxido-reductase).
See the text for details. FMN, flavin mononucleotide; FeS, iron–sulfur complex; Q, oxidized quinone or ubiquinone; QH2, reduced
quinone; QH, semiquinone; cyt c, cytochrome c; CuA and CuB, copper complexes A and B; His, histidine; IMS, intermembrane
space. (B) Structures of quinones (left) and heme. Structures taken from the KEGG database (http://www.genome.jp/kegg/kegg2.
html): oxidized ubiquinone C00472, reduced ubiquinol C00530, and Fe-heme C00032. (C) High-resolution structures of bacterial
NADH:quinone oxido-reductase (complex I; PDB accession 3M9S) from Thermus thermophilus, and cytochrome bc1 reductase
with antimycin inhibitor (complex III; PDB accession 1PP9) and the reduced mitochondrial cytochrome oxidase (complex IV; PDB
accession 1V55), both from cow mitochondria.
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341
BOX 9.1 Electron-transport chains as proton pumps (continued)
There, the quinol is oxidized and the two protons are
released from the membrane, while the two electrons are
captured by heme binding sites—one electron is located
to the peripheral membrane protein cytochrome c to
be transferred to complex IV, while the other is recycled
within complex III to a quinone that is reduced to a semiquinone (QH), while capturing an H+ from the matrix
side. A second recycled electron will capture a second H+
and the fully reduced quinol (QH2) will join the quinone
pool. Thus, two reduced quinones are able to pump four
protons across the membrane. The mechanism of proton pumping by this Q-cycle depends on the interplay
of hydrogen and electron-only accepting units; that is,
ubiquinone for hydrogens, and FeS and cytochrome
hemes for electron-only binding sites (Figure 1B).
The cytochrome c shuttle connects the electron flow
from complex III to complex IV (the cytochrome c oxidase), which absorbs the shuttled electron onto a copper binding site (CuA). Complex IV pumps protons via a
histidine binding site that cyclically changes in its protonation state in response to the redox state of the nearby
binuclear CuB–heme center, where oxygen is sequentially reduced to water. Complex IV moves electrons
from the CuA site via a series of two hemes to a second
copper binding site. This latter site also binds oxygen and
protons, facilitating the reduction of oxygen to water. For
each molecular oxygen (O2) being reduced, four protons and electrons are consumed to form two molecules
of water. The electron pathway across complex IV, from
the cytochrome-binding site to the water-forming site,
is coupled to a proton pump that shuffles an additional
four protons out of the matrix compartment for each O2
used. This pump is based on an electrostatic proton-wire
pathway. Proton movement compensates the negative
charge imbalance caused by electron flow with positively charged hydrogen ions.
Photosynthesis also uses an electron-transport chain to
pump protons (Figure 2). However, photosynthesis uses
an electron donor whose electrons do not have enough
energy to do work. Hence, the absorption of light energy
is used to activate those electrons. In plants, algae,
and cyanobacteria, the electrons flow through a linear
electron-transport chain that both produces a proton
gradient to make ATP and synthesizes the high-energy
metabolite NADPH/H+. In this linear electron-transport
chain, electrons come from water molecules that are
oxidized by the oxygen-evolving complex in photosystem II (PSII). Water oxidation releases four protons and
four electrons from two water molecules, reducing four
manganese (Mn) ions from an MnIV to MnIII state in the
oxygen-evolving complex. The electrons are then loaded
one at a time onto a chlorophyll molecule for photon
plants, cyanobacteria; linear
H2O
hν
THYLAKOID
LUMEN
e–
4Mn
O2 H+
+
H+ H+ H
H+
H+ H+
e–
PC
P680
PSII
STROMA
P700
e–
cytochrome b6f
e–
PERIPLASM
e–
PSI
PQH2
PQH2
PQ e–
PQ
H+
H+
H+
CYTOPLASM
Fd
NADPH +/H+
purple non-sulfur proteobacteria; circular
Figure 2 Linear and cyclic light reactions. Photosynthesis in plants is a linear electron-transport chain extracting electrons
and H+ from water on photosystem II (PSII). Light absorption activates the electron bound to the special-pair chlorophylls
(P680, or pigment with maximum spectral absorption at 680 nm). The activated electron feeds a proton pump maintained by
the plastoquinone (PQ) pool and cytochrome bf complex. Having low energy levels at this point, electrons are transported
to photosystem I (PSI) via plastocyanin (PC; a Cu2+ binding site), where a second round of photon activation occurs (P700
chlorophyll special pair). The activated electrons are funneled via ferredoxin (Fd) and NADP reductase to capture the highenergy hydrogen. In a cyclic mode, photosystem I shuttles the electron back via ferredoxin to the plastoquinone pool for proton
pumping. No further electron supply from water is needed in the cyclic mode. The latter mode is predominant in purple non-sulfur
proteobacteria (for example, Rhodobacter sphaeroides and Rhodopseudomonas spp.) and has also been observed in plants
under low light conditions.
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Chapter 9: Metabolism: Growing Membranes, ­Sustaining Cells
BOX 9.1 Electron-transport chains as proton pumps (continued)
capture. As the oxygen-evolving complex is located in
the thylakoid lumen, the released protons accumulate
without pumping (no membrane transport is needed)
and are a primary source of protons used by the ATP
synthases clustered on the stroma surface of the thylakoid stacks and the stroma lamellae. Additional protons
are pumped from the stroma into the thylakoid lumen
by the plastoquinone pool and the cytochrome bf complexes. The process is analogous to the mitochondrial
Q-cycle proton-pumping mechanism of complex III (the
cytochrome bc1 complex). The electrons are then shuttled via plastocyanin (a cytochrome c analog) to photosystem I (PSI), where they are activated again by photons
in a special-pair chlorophyll complex (P700). From there,
activated electrons are funneled on the stromal surface
via ferredoxin and NADP reductase to NADP+ in order
to make the high-energy compound NADPH/H+ in the
presence of protons.
Some photosynthetic organisms do not have a photosystem II and operate in a cyclic transport mode, as found
in purple non-sulfur bacteria. These bacteria cycle electrons back-and-forth between PSI (light activation) and
the cytochrome bf complexes feeding the proton pump.
To operate in a cyclic fashion, ferredoxin reduces plastoquinones instead of NADP+; this shuttles activated
electrons, which pick up an H+ on the stromal side, back
to the cytochrome bf complex to start another round of
proton pumping. Electrons are not used up in this cyclic
reaction scheme and no water is needed. Since this process does not produce NADPH/H+, no carbon fixation
can occur, but lots of ATP is synthesized.
The respirasome explains how membrane protein
complexes form functional units
The electron-transport chains in respiration and photosynthesis are good
examples of how enzyme complexes involved in a tightly controlled metabolic pathway are not only working together functionally but are closely
associated into supercomplexes, allowing for efficient substrate channeling. The respiratory electron-transport chain is composed of four large
protein complexes (I–IV), three of which form a functional unit—the
proton-pumping respirasome (complexes I/III/IV) (Figure 9.12A). The
respirasome generates a proton gradient which in turn powers the ATP
synthase. The combined process is known as oxidative phosphorylation
and is the main ATP-synthesizing pathway in mitochondria and aerobic
prokaryotes. The respirasome is a supercomplex made of complexes I, III,
and IV that efficiently couples the NADH oxidation to proton pumping (see
Box 9.1, Figure 1). The close association of all three complexes minimizes
the distance required for the quinone pool and cytochrome c shuttle to
carry their load between complexes; the pathways for these small carrier
molecules are now extremely short and do not depend on long, random
motion in the core of the lipid bilayer. The association between these complexes appears not to be fixed, however, and alternative associations, such
as complex I/III and III/IV dimers, have been observed. The latter works in
conjunction with complex II, which directly links the Krebs cycle to oxidative phosphorylation via its FAD co-factor (complex II is actually succinate
dehydrogenase). It is the only complex of the electron-transport chain that
is not a proton pump.
The organization of the electron-transport chain in mitochondria is also
a good example to illustrate compartmentation involving membranes
(Figure 9.12B). Oxidative phosphorylation (electron-transport chain
and ATP synthase) is organized within the cristae infoldings of the inner
membrane of the mitochondrion rather than at the smooth surface location where the inner and outer membranes are in close proximity. As a
result, the narrow space between cristae membranes—topologically, an
extension of the intermembrane space between the outer and the inner
membranes—can be considered its own metabolic compartment (a
three-compartment model) where protons accumulate as a result of the
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(A)
respiratory-chain complexes
respirasome
IV
I
(B)
II
outer
membrane
III
inner
membrane
II
ATP
synthase
I/III/IV
(C)
2 compartments
H+
ATP
1
3
O2 H+
H+
3 compartments
1
TCA
H2O
ATP
synthase
2
2
NADH
H+ H+
H+ H+ H+
H+
mitochondria
mitochondrial network
ADP+Pi
mitochondrial matrix
electron-transport chain and feed directly into the ATP synthase (Figure
9.12C). Thus, protons are unlikely to escape into the cytosolic compartment. The intermembrane space between the inner and outer membranes
of the mitochondrial envelope (compartment 1 in the three-compartment
model shown in Figure 9.12C) is reserved for transport and biosynthetic
and degradative pathways. Various di- and tricarboxylic acids and the
ADP/ATP antiporters in the inner membrane (see Table 9.1) provide the
exchange of metabolites between the matrix compartment and the intermembrane space, which in turn is connected via large porin channels
(voltage-dependent anion channels; VDACs) to the cytosol. Finally, what
constitutes a mitochondrion varies from cell to cell, or within cells, as they
can be organized into multiple, small, dissociated organelles or branched
reticulocyte networks. The take-home message is that there is a dynamic
aspect at all levels of organelle organization and thus compartmentation,
from protein-complex formation (respirasome organization) to luminal
space topology and even whole organelle structure.
343
Figure 9.12 Mitochondrial metabolic
compartmentation. (A) The organization
of the respirasome. The respiratory-chain
complexes I through V form a functional
unit comprising the electron-transport
chain (complexes I through IV) and the ATP
synthase, complex V. However, biochemical
evidence shows that complexes I, III, and
IV form a supercomplex known as the
respirasome. (B) Oxidative phosphorylation
is organized within the cristae lamella of
the inner membrane of mitochondria. The
space between adjacent inner membranes
serves as the proton-gradient reservoir,
allowing for an efficient coupling of the
electron-transport chain proton pump with
the ATP synthase complex. The tricarboxylic
acid (TCA) or Krebs cycle feeds reducing
equivalents (e–/H+) into the respirasome
via NADH. Substrates and products of
respiration are transported across the outer
membrane via voltage-dependent anion
channels (VDACs) or porins and across
the inner membrane via corresponding
mitochondrial transporters (see Table 9.1
for details). (C) Metabolic compartmentation
in mitochondria is dynamic and occurs
at multiple hierarchical levels. Within the
inner membrane, the respiratory complexes
are organized into the respirasome and
complex II (see (A)). At the organelle level,
the intermembrane space can be further
compartmentalized into the space between
adjacent inner and outer membranes, and
also within cavities enclosed by cristae
folds. Lastly, mitochondria can exist as
multiple individual organelles or fuse to
form a single branched reticulocyte or
membrane network. (Adapted from Brière
J-J, Chrétien D, Bénit P & Rustin P [2004]
Biochim Biophys Acta 1659:172–177. With
permission from Elsevier.)
Thylakoid membranes compartmentalize light-harvesting
megacomplexes
The electron-transport chains of both respiration and photosynthesis are
related in several ways. Both are proton pumps that use a quinone pool and
a cytochrome bc or bf complex, respectively, to create a proton motive force
suitable for ATP synthesis. Both are organized into specialized membrane
folds: cristae in mitochondria and thylakoid membranes in chloroplasts. The
thylakoid membranes themselves are organized into grana stacks and stromal
lamellae, which promotes lateral compartmentation of the photosynthetic
electron-transport chain, which is driven by molecular interactions between
proteins, and between proteins and lipids, as well as by changes in tension due
to local curvature (Figure 9.13). The membrane stacks (grana) are enriched
with photosystem II (PSII) and their surrounding light-harvesting complex
(LHCII) units. PSII contains the oxygen-evolving complex that extracts hydrogens from water leaving behind molecular oxygen as a waste product. The
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Chapter 9: Metabolism: Growing Membranes, ­Sustaining Cells
(A)
grana stacks
stroma lamellae
(B)
PSI
S
PSII–LHCII
supercomplexes
S
ATP synthase
cyt b6/f
Figure 9.13 Structural organization
in thylakoid stacks. (A) Thylakoid
membranes are organized into grana
stacks and stroma lamellae. Grana
stacks contain photosystem II (PSII) and
the associated light-harvesting complex
II (LHCII) as well as the cytochrome
bf (cyt b6f) complex. Stroma lamellae
contain mostly photosystem I (PSI) and
associated light-harvesting complexes I
and cytochrome bf complexes. The ATP
synthase is distributed along the lamella
and stromal surfaces of the grana stacks.
(B) Model of a grana stack (top) and
connecting stromal lamella (S) as derived
from freeze-fracture electron micrographs
(bottom; arrows show stacked thylakoids).
The model shows how each fold of the
grana stack is connected to the lamellae.
(Adapted from Dekker JP & Boekema EJ
[2005] Biochim Biophys Acta 1706:12–39.
With permission from Elsevier Inc.)
50 nm
stromal lamellae and grana surfaces contain photosystem I (PSI) and its associated light-harvesting complexes (LHCI) as well as ATP synthases. The protonpumping machinery—that is, the cytochrome bf complexes—is distributed
evenly between the grana and the lamellae membrane compartments.
The amassing of light-harvesting complexes and PSII makes grana stacks
efficient photon-capturing structures. Referred to as antenna complexes, the
light-harvesting complexes transfer resonance energy (but not electrons) to
chlorophylls (P680) of the centrally located PSII, where the electron-transport chain is actually initiated (see Box 9.1, Figure 2). The compartmentation
of PSII and PSI complexes into different membrane areas of the thylakoids is
also thought to benefit the control and efficiency of photosynthesis in chloroplasts. The compact stacking of PSII and light-harvesting complexes into
the grana improves light absorption under limited light availability. In addition, physical separation of PSI and PSII is thought to prevent bypassing of
the cytochrome bf proton pump by an uncontrolled flow of electron energy
from the PSII light-harvesting complexes directly to PSI complexes, which
would reduce the efficiency of the proton motive force. Finally, separation
of these two photosystems allows chloroplasts to easily switch from linear
electron transport to a cyclic transport mode. The latter mode cycles electrons back and forth between PSI (light activation) and the cytochrome bf
complexes feeding the proton pump. Because of the lateral compartmentation of PSII into grana and PSI into lamellae membranes, the cyclic mode is
restricted to the stromal lamellae only.
Building Membranes, Building Cells
Modern membranes are self-renewing structures and are
not built from scratch
In 1855, the German physiologist Rudolf Virchow postulated that “all living cells arise from pre-existing cells.” Virchow was expanding on the then
twenty-year-old cell theory by the botanist Matthias J. Schleiden and the
zoologist Theodor Schwann that “all living things are made of cells” and
that cells form the “basic units of structure and function in living things.”
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With evolution on his mind, Virchow challenged the contemporary view of
the spontaneous generation of life, a few years before Charles Darwin published his theory of evolution in 1859 and before Louis Pasteur showed in
1862 that sterilized media did not grow new bacteria, unless exposed to air.
The evolutionary biologist August Weissman summed it up in 1880 by saying
“that cells living today can trace their ancestry back to ancient times.” He was
alluding to the existence of a common ancestral cell. How this ancestral cell
came into being is an entirely different and largely unknown story.
What is true for cells seems to be true for their membranes. There is no evidence that cell membranes are made from scratch, but that they arise from
growth of preexisting cell membranes followed by fission (cell division) or
fusion (fertilization). Thus, the modern view of cell membranes is that of
self-renewing structures whose formation depends on the growth of already
existing membranes (Figure 9.14). Extending self-renewal into the past, one
can postulate that all modern cell membranes share a common ancestral
membrane (see also Figure 1.1).
Instead of growing through self-assembly, modern membranes grow by synthesizing new lipids and proteins into specialized preexisting membranes
and, in compartmentalized cells, distributing them as vesicular membranes
to other membranes. Lipid synthesis starts with lipid building blocks (for
example, palmitic acid, phosphoglycerols, sphingosine, mevalonate, or
monosaccharides) made in the cytoplasm (see Figure 9.2). Modifier enzymes
in the smooth ER membrane (or plasma membrane in prokaryotes) catalyze
elongation, desaturation, cyclization, and oxygenation to produce various
forms of fatty acid chains, sterols, or hopenes, which are then combined into
phospholipids and glycolipids. After synthesis of basic membrane lipids
(phosphatidic acid, ceramide, lipid A, or sterol), modifications of headgroup structures are introduced by additional membrane-bound enzymes.
345
SELF-ASSEMBLY
first membrane
synthetic membrane
SELF-RENEWAL
modern cell
membrane
Figure 9.14 Formation of new cell
membranes. The formation of cell
membranes comes in two forms: from lipid
and protein self-assembly and from already
existing cells through cell (or organelle)
division. Self-assembly is not found in
living cells but is routinely used to make
artificial membranes in the laboratory (see
Box 5.4) and is the current working model
for the primordial evolution of the first cell
membrane needed to form a protocell.
Modern cell membranes come from already
existing membranes, a process best called
self-renewal—the incorporation of new lipid
and protein building blocks into preexisting
cell membranes, which grow and duplicate
by membrane fission.
As membranes grow in size, they can release new, independent membranes
through cell division (fission) or by budding off small vesicles that fuse with
and maintain the multiple internal organellar membranes in eukaryotic
cells, except for the mitochondrial and chloroplast membranes. These latter
two membranes grow and divide independently, although they must acquire
most of their membrane components from the main cell; that is, they absorb
ER-made lipids through a protein-based shuttle system and have their own
protein-import machinery (see Figure 7.35). What cells do not do is synthesize their lipids first and then let them self-assemble from micelles into vesicles, a common strategy of forming synthetic membranes in a test tube (see
Boxes 5.1 and 5.4).
The first membranes were built differently
While biologists agree that no new life is created from building blocks through
self-assembly today, they also agree that such self-assembly must have been
instrumental at the dawn of life some three to four billion years ago. The study
of the origin of life, its prebiotic origin, focuses on three separate issues: first,
the formation of a metabolism to extract energy from the environment for
growth, movement, and reproduction; second, the creation of self-copying
polymers that function as genetic material (memory) with built-in catalytic
activity; and third, the formation of an envelope or membrane that separates
this self-replicator from the environment. Together, the membrane, the metabolic network, and the replicator form the first form of cellular life.
Models of the emergence of the first membrane are all based on self-assembly properties (discussed in Chapter 5) of surface-active substances like fatty
acids, fatty alcohols, and prenols. Experiments with oleic acid and shorterchain fatty acids show that these simple lipids form stable vesicular compartments withstanding osmotic stress induced by a glucose gradient with
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Chapter 9: Metabolism: Growing Membranes, ­Sustaining Cells
tension up to 10 mN/m in experiments simulating prebiotic conditions. The
comparably stronger bilayers made of phospholipids hold up to 25 mN/m
of osmotic pressure. Many models of prebiotic life suggest that compartmentalization of metabolic and genetic elements started on clay surfaces or
within other porous solid materials that prevent osmotic rupture, only to be
released later as free-living cells with lipid membranes and transport systems. The true origin of any first form of life is not known.
A membrane can be built in the lab as if it
were the first one
Ideas of self-assembling lipids that can spontaneously form membranes is
not mere fantasy, but has its basis in experimental evidence, namely the
ability of phospholipids (and some fatty acids) to form synthetic membranes
in test tubes, including liposomes, vesicles, monolayers, and planar bilayers.
Extremely simple procedures of mixing lipids with water prove that many
amphipathic molecules aggregate in a nonrandom fashion leading to supramolecular assemblages with new emerging properties (see Chapter 5). This
self-assembly property of amphipathic molecules in water demonstrates
the formation of complex entities from simpler building blocks. While lipid
self-assembly is not a proper model to explain self-renewal of biological cell
membranes, self-assembly in cells is still thought to be critical on a smaller
scale for protein folding and protein-complex formation. Examples include
formation of cytoskeletal fibers or protein coats of viruses, or in cell–cell
junctions of membranes, receptor clustering, or respirasome formation.
The formation of lipid domains is also an example of self-assembly in the
broader sense of phase separation.
Much of what we know about self-assembly processes comes from reductionist analysis of membrane components and their careful reconstitution
into model membrane systems (see Box 5.4). A very interesting example of
such reconstitution is the formation of synthetic proteoliposomes or vesicles
that can synthesize ATP in the presence of light (Figure 9.15). These artificial photosynthetic membranes are made of three components only: a lipid
bilayer made of synthetic phosphatidylcholines, a mitochondrial F1FO-ATP
synthase purified from bovine liver, and bacteriorhodopsin, a light-driven
Figure 9.15 Constructing an artificial
photosynthetic cell. (A) This synthetic
cell is a proteoliposome (artificial vesicle)
constructed in vitro from synthetic
phospholipids (phosphatidylcholines),
bacteriorhodopsin, and bovine
mitochondrial F1FO-ATP synthase. Upon
light activation of bacteriorhodopsin,
protons accumulate in the vesicle lumen
and can drive the ATP synthase by
chemiosmosis. (B) Ribbon diagram of
bacteriorhodopsin, a light-driven proton
pump, from the archaeon Halobacterium
salinarum. The structure is derived from
the Protein Data Bank entry 2BRD. (C)
The composite structure of an F1FO-ATP
synthase. The F1 structure of ATP synthase
is derived from the bovine mitochondrial
protein and solved by X-ray diffraction
analysis (PDB entry 2WSS). The c subunit
structure of the FO part comes from the
E. coli ATP synthase through nuclear
magnetic resonance solution-structure
analysis (PDB entry 1C0V).
CellMembranes ch09.indd 346
(A)
(B)
H+
hν
(C)
N
synthetic
phospholipids
C
H+
H+
H+
bacteriorhodopsin
+
H+ H
H+
H+
H+
H+
ADP + Pi
bovine F1FO-ATP synthase
ATP
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Building Membranes, Building Cells
347
prokaryotic proton pump purified from the archaeon Halobacterium salinarum. The in vitro reconstitution of a fully functional membrane system not
only demonstrates the ability of select solutes to self-assemble, it also shows
that biomolecules of widely different origin—synthetic lipids plus proteins
extracted from a eukaryotic organelle and an extremophile archaeon—
readily work together as a fully functional unit. This is consistent with the
model of a common ancestor that all modern forms of life share, and that
the apparent diversity of all modern organisms has an underlying molecular
unity. That is to say, humans, viruses, bacteria, fungi, protozoans, and plants,
we are all made of the same building blocks.
The proteoliposomes used in such reconstitution experiments are commonly formed through a simple process of detergent dialysis. Solutions of
detergent, lipids, and proteins are put into small dialysis bags and left for several hours in a large volume of buffer solution. The porous membrane of the
dialysis bag allows diffusion of monomeric molecules, but not micelles, into
the dialysis buffer compartment. It is mostly detergent molecules that are in
free equilibrium between the mixed micelle and monomer detergent phase.
As these free detergent molecules diffuse out of the dialysis bag, the lipids
and proteins trapped in the micelles get depleted and eventually aggregate
to form liposomes with proteins incorporated into the vesicular bilayers.
Because of the randomness of the liposome formation, the transbilayer orientation of both the lipids and membrane proteins is not controlled and is
thus random. This distinction is of course important for transport proteins,
leaving some vesicles functionally oriented outside-in and others insideout. Proteoliposomes with correctly oriented proteins can be selected by the
degree of liposome destabilization and the properties of the reconstituted
proteins. All three properties have an impact, making it possible to get >90%
of proteoliposomes as right-side in. In the absence of structural orientation,
adding inhibitors to the inside or outside of the vesicle compartment creates
functional asymmetry suitable for experimental analysis, by inactivating all
those proteins incorporated in the ‘wrong’ direction.
Internal membranes promote the evolution of larger cells
There is an intriguing aspect of cellular life: the existence of only two basic
cell types—prokaryotic and eukaryotic—despite billions of years of evolution. These cell types are organized very differently, yet share many similarities. The most striking difference is that prokaryotic cells are small compared
to eukaryotic cells and operate with only one metabolic compartment and a
single membrane, the plasma membrane (with the exceptions of the Gramnegative and anammox bacteria already discussed). Prokaryotic plasma
membranes carry out all transport functions, membrane synthesis, energy
production, signaling, adhesion, and motility (Figure 9.16). In eukaryotes,
membrane synthesis and energy production are carried out exclusively by
membranes of organelles, which frees-up their cell surface area for transport, adhesion, and signaling.
The various complex membrane structures of organelles show that having
multiple membranes is not just a matter of a division of labor, but an advantageous structural feature for cells to grow in size and form multicellular structures. Let’s look at size first. Scaling-up cells into larger ones means running
into energy supply problems. Of all metabolic activity, the ability to produce
large amounts of ATP comes from membranes. Thus, if a cell gets most of its
available energy from membrane-bound processes, the membrane area will
be the limiting factor in how much energy a cell can produce as it grows in size.
The reason for this limitation is based on the observation that for a growing
cell, surface area and volume increase geometrically but at different rates. The
surface area increases to the power of two (x2) while the volume increases to
the power of three (x3); that is, the cell’s internal volume increases faster than
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Chapter 9: Metabolism: Growing Membranes, ­Sustaining Cells
Figure 9.16 Functional specialization
of internal membranes in eukaryotic
cells. (A) All membrane functions in
prokaryotic cells—synthesis, transport,
energy production, signaling, adhesion,
and motility—are carried out by the
cytoplasmic membrane. Adhesion is
mediated by fimbriae and pili, while motility
is provided by rotary flagella. These types
of protein filaments are anchored in the
cell membranes. The outer membrane
of Gram-negative bacteria is integral
to the cell wall structure, plays a role in
protection, has supportive roles in transport
as a generic molecular sieve, and plays a
part in recognition and adhesion. (B) The
membranes of eukaryotic cells specialize
in various activities. All membranes
are involved in transport to provide the
needed metabolites to the corresponding
compartments. The plasma membrane
specializes in signaling, recognition,
motility, and adhesion. Mitochondrial and
chloroplast membranes carry out energy
metabolism. The nuclear–endomembrane
system is responsible for storage and
retrieval of genetic information, the
biosynthesis of membrane components
and secretory pathway activity, the
distribution of cellular components, and
the degradation of metabolites and cell
structures.
(A)
Figure 9.17 How to increase the
metabolically available membrane area
in larger cells. (A) When a cell increases in
size by a certain proportion (for example, its
length X doubles), the relationship between
the volume of the cell and its surface is
governed by a power law, the square-cube
law. The volume of the cell increases to
the power of three (cubic; 23 = 8), while
the cell surface area increases only to the
power of two (square; 22 = 4). As a result,
larger cells have proportionally less surface
area per volume than smaller cells. (B) To
increase the metabolic capacity of their
membranes to sustain the faster-growing
cell volume, larger cells contain multiple
internal membrane systems—that is,
organelles that increase the available
membrane area without increasing the
already larger cell volume. (C) An additional
increase in membrane surface area without
increasing the cell size can be achieved by
having internally folded membranes. This is
found in mitochondria and chloroplasts and
a few select prokaryotes. (D) Geometry is
important, however. Prokaryotes minimize
the progressive decrease in surface area
to volume ratio by growing elongated,
rod-shaped cells rather than spherical
structures, and eukaryotic cells can grow
long yet thin tubular dendrites and axons.
(E) Instead of growing a larger cell to
increase the size of an organism, multiple
smaller cells form a larger organism.
Now we are in the position to make the case that compartmentalization and
use of internal membranes was a likely prerequisite for the evolution of larger
cells and multicellular organisms. By shifting the membrane-area-hungry
energy-production processes of respiration and photosynthesis from the
surface of the cell to its inside, eukaryotic cells can maintain a high energy
output while maximizing cell surface signaling and interactions with other
cells (Figure 9.17B). But simply adding organellar membranes did not seem
enough to supply eukaryotic cells with energy. Mitochondria and chloroplasts have also increased their energy-producing membrane surface areas
CellMembranes ch09.indd 348
(B)
transport
adhesion
lysosome
energy
degradation
motility
energy
synthesis
synthesis
nucleus
mitochondrion
peroxisome
Golgi
adhesion
motility
smooth
ER
rough ER
chloroplast
energy
transport
synthesis
signaling
vacuole
storage and
degradation
synthesis
endosome
signaling
its surface area (Figure 9.17). For a cell that doubles in size (radius r or side
length a), the membrane surface area increases fourfold, while the volume
increases ninefold. As a result, the surface area to volume ratio decreases with
increasing cell size (1/x) and the functional capacity of the membrane lags
further and further behind the metabolic demand of the cell interior as the
cell grows. The cell membrane is less and less able to supply enough energy
and distribute the necessary building blocks as it grows ever larger.
(A)
(B)
(C)
(D)
(E)
cell volume
increase in area and volume
membrane area
fV(X3)
100
50
10
fA(X2)
0
1
2
3
4
factor of cell increase (X)
5
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Building Membranes, Building Cells
349
by folding (cristae) and stacking (grana) (Figure 9.17C). Very few prokaryotes
have come across this solution to increase their energy output, likely because
they do not need to do so, as their cells stayed small, but also because their
cytoplasmic volume would be reduced, leaving them with less space for their
chromosome, ribosomes, and metabolic enzyme machineries.
Membrane heredity maintains membrane structural and
functional diversity
Having internal membranes to carry out specialized functions means that a
eukaryotic cell has to make and maintain membranes with unique characteristics, compositions, functions, and physical abilities. Most membranes of
eukaryotic cells are separate from each other but made and maintained from
a single pool of newly synthesized lipids and proteins via membrane trafficking, docking complexes, and transfer proteins (nonvesicular transfer). The
nuclear–endoplasmic membrane system is the site of most lipid (and most
membrane protein) biosynthesis (mitochondria and chloroplasts have their
own sets of modifier enzymes as discussed below). We say that the ER membrane is a feeder or genetic membrane (Figure 9.18). It is able to self-renew
and replenish the endosomal–lysosomal membrane system. Plasma membranes, endosomes, lysosomes, vacuoles, peroxisomes, and the Golgi stack
are all derived from this feeder membrane by vesicle transport and recycling. Deprived of its endosomal membrane system, the plasma membrane
could neither grow nor change.
Mitochondria and chloroplasts, as well as many plastids in plants, fungi, and
protists, are different. They form their own genetic lineages and can only be
derived from themselves, although they lack some enzymatic machinery to
be self-sufficient and do not synthesize all of their own membrane components. For instance, mitochondrial lipids are derived from precursor lipids
(for example, phosphatidic acid) synthesized in the ER and transported
from the ER to the organelle’s outer membrane via mitochondria-associated membrane junctions. There, organelle-resident enzymes modify lipid
head groups to the specific functional needs of the organellar membranes.
Prokaryotes typically have only one membrane system, the plasma membrane, although Gram-negative bacteria are an exception, having a second,
derived outer membrane. This second membrane receives its lipid and proteins through protein transfer systems.
(A)
PROKARYOTES
outer
membrane
inner
membrane
plasma
membrane
EUKARYOTES
nucleus/ER
Golgi
endosome/
lysosome
mitochondrion
chloroplast
(B) cyanobacteria
archaea
α-proteobacteria
endosymbiotic
membranes
1
2
1
chloroplast
mitochondrion
2
nuclear–endosomal membranes
The use of organellar energy-producing membranes is likely the reason for
a second benefit, the evolution of multicellularity. Using internal membranes for a process demanding a large membrane area frees the plasma
membrane to devote more of its surface to transport, signaling, and, most
consequentially, cell adhesion. Increased ability and diversity of signaling
and adhesion in particular are central to integrating and coordinating cellular activity in multicellular organisms. It is thus likely not a coincidence that
multicellular organisms have evolved exclusively in the domain Eukarya.
host
3
ER
nucleus
3
Golgi
lysosome,
peroxisomes
plasma membrane
Figure 9.18 Membrane inheritance—genetic and derived membranes. (A) There are four major lineages of genetic membranes
that act as feeder membranes for other derived membranes: (i) prokaryotic membranes; (ii) the eukaryotic nuclear–endoplasmic
reticulum (ER) membrane system; (iii) mitochondrial membranes; and (iv) chloroplast membranes. Genetic or feeder membranes
cannot be derived from any other membrane lineage, and can never be lost. For instance, within a eukaryotic cell, a mitochondrion
can only come from a mitochondrion, and not from the nuclear–endosomal membrane system, although most mitochondrial lipids are
derived from precursor lipids imported from the endoplasmic membrane system (dashed arrows). The same considerations hold true
for chloroplast inheritance. In prokaryotes, the site of membrane synthesis is the plasma membrane, which is the inner membrane
in Gram-negative bacteria. The outer membrane of these bacteria is a derived membrane and its components are synthesized
via the inner membrane (plasma membrane) using protein transport systems across the periplasmic space. (B) The origin of the
different genetic membrane lineages within eukaryotic cells is consistent with the endosymbiotic theory of the origin of mitochondria
(1) from ancient free-living α-proteobacteria and of chloroplasts (2) from ancient photosynthetic cyanobacteria. The folded cristae
and additional thylakoid membranes appear to have evolved after symbiosis occurred. The cell membrane and nuclear–endosomal
membrane system are thought to originate from an archaeal ancestor (3) with already infolded or internal membranes.
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The term ‘genetic membrane’ was coined by the evolutionary biologist
Thomas Cavalier-Smith to allude to the fact that there is more than one type
of self-renewing membrane and that some membranes are derived rather
than being able to self-renew. The term ‘genetic membrane’ does not just
imply that they are self-renewing, but also that once lost, they cannot be
derived from another membrane lineage. The term ‘genetic membrane’ thus
embraces the idea of inheritance, taking into account that (i) membranes
are the offspring of evolutionarily independent lineages, basically tracing
the three domains of life (Archaea, Bacteria, and Eukarya), and (ii) they are
able to self-renew but cannot be derived from any other membrane lineage.
Consistent with the endosymbiotic theory of the origin of energy-producing
organelles in eukaryotes, the mitochondrial membranes are the descendants of primitive α-proteobacteria, while the chloroplast membrane systems
are derived evolutionarily from an ancient endosymbiotic cyanobacterium. As a consequence of the almost complete reduction of their ancestral
genomes to small, modern organellar chromosomes, both organelles largely
depend on the nuclear chromosome for the encoding and synthesis of most
of their lipid and membrane proteins. Both organelles, although self-renewing, depend on the supply of both lipids and proteins from the ER feedermembrane system.
Spatial organization is important for membrane renewal
The information defining what a cell is lies in its three-dimensional organization and capacity to self-renew its membranes and subcellular compartments. The genome does not contain this spatial information. One cannot
put a chromosome into a soup of cellular components and expect it to build
a cell. Once the cell is damaged, it will be irretrievably destroyed. To understand this point better, one has to distinguish the template-based synthesis
of nucleic acids and proteins from the way that carbohydrates, lipids, and
protein complexes (for example, cytoskeletal filaments, adhesion junctions, and the respirasome) are synthesized. Their structure is not encoded
in a linear template that determines the final sequence/structure (see also
Figure 4.3). Instead, cells depend on their highly organized spatial compartmentalization as the basis of the biosynthesis of complex structures. For
instance, the synthesis of oligosaccharide moieties of glycolipids and glycoproteins operates much like an assembly line and is encoded by sorting tags
that say ‘if you carry this signal, you go there’ where further modification
and distribution can occur. The enzymes along the assembly line must be in
proper spatial order from the site of origin of synthesis to where the finished
product is actually used.
No matter what form or mechanism the membrane renewal takes, the cell
needs to conserve three membrane characteristics during synthesis: the
type of membrane (genetic origin) and its composition; the polarity and orientation of the components (for example, facing the cytoplasmic or extracellular/luminal side); and, in double-membrane organelles and prokaryotes,
the topological location relative to other membranes (for example, outer
and inner membranes). The secretory pathway in eukaryotic cells perfectly
reflects the significance of this three-dimensional assembly line that orchestrates the highly conserved synthesis of cell membranes. It connects the
feeder ER membrane to its derived endomembrane organelles (nucleus,
Golgi, lysosomes, endosomes, vacuole, and peroxisomes) and the plasma
membrane (Figure 9.19A). While transport between various membrane
compartments along the secretory pathway is achieved by vesicle transport, eukaryotic cells also use an extensive network of lipid-transport proteins between the ER and mitochondria and chloroplasts and, selectively,
within the nuclear–endosomal membrane system. For instance, a nonvesicular lipid-transfer mechanism exists between the ER and trans Golgi
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351
membranes (Figure 9.19B). The ceramide transport protein CERT is a cytosolic (water-soluble) protein that extracts ceramide from the ER membrane
and transports it by a nonvesicular mechanism to the Golgi. Thus, a parallel mechanism to vesicle-based trafficking exists. It is thought that CERT
can either move from an ER to a trans Golgi membrane, or form a bridge
between adjacent ER and trans Golgi membranes.
Similarly, phospholipid transport is commonly found at junctions between
the ER and mitochondria (and plastids in plant cells) made by a tetrameric
tethering complex composed of proteins Mmm1, Mdm10, Mdm12, and
Mdm34. These proteins are named after their role in maintaining mitochondrial morphology and distribution. Additional lipid-transfer proteins, such
as the intermembrane space protein Ups1 in yeast, shuttle phosphatidic
acid (PA) from the mitochondrial outer membrane to the inner membrane,
where PA is converted to phosphatidylglycerol and cardiolipin or diphosphatidylglycerol (DPG), the two major mitochondrial inner-membrane
phospholipids. There is no known vesicle-based lipid-exchange mechanism
between the ER and mitochondrial or plastid outer membranes.
Likewise, the double-membrane cell envelopes of Gram-negative bacteria employ lipid (and protein) shuttle systems to build outer membranes.
Lipopolysaccharide (LPS) units are fully synthesized in the inner membrane
(A) VESICULAR TRANSPORT
plasma
membrane
(B) NONVESICULAR TRANSPORT
EUKARYOTES
endosome/
lysosome
PROKARYOTES
mitochondrion
secretory
vesicle
mitochondrion
CYTOPLASM
Golgi
apparatus
peroxisome
Mdm
PC
LPS
IM
ER
trans Golgi
OM
chloroplast
nucleus
vacuole
endoplasmic
reticulum
OM
Lpt
complex
CERT
ceramide
ER
lipoprotein
Lol
shuttle
IM
Figure 9.19 Lipid transfer between membranes. (A) Classical trafficking routes between the nuclear–endosomal membrane
system and the plasma membrane in eukaryotic cells use vesicle transport for both lipids and proteins. Vesicular trafficking connects
the nuclear–endoplasmic reticulum (ER) membrane system (feeder membrane) to the plasma membrane via the Golgi and the
endosomal–lysosomal sorting and degradation apparatus. Part of the connection between the organellar membrane network and
the plasma membrane is exocytosis and endocytosis coupling the secretory pathway and vesicle recycling. Membrane trafficking
moves lipids and proteins among the various membranes, yet organelles retain certain lipids and proteins to maintain their unique
compositions and functions, while allowing others to be passed on to the next compartment. Vesicle trafficking is not known to exist
between the ER and mitochondria and chloroplasts/plastids. (B) Nonvesicular lipid transport connects the ER and mitochondrial outer
membrane at contact points (junctions or mitochondria-associated membranes [MAM]) containing proteins known as ‘maintenance of
mitochondrial morphology’ and ‘distribution’ (Mdm and Mmm). These junctions transfer mostly phosphatidylcholine (PC), phosphatidic
acid (PA), and phosphatidylserine phospholipids. PA is shuffled to the inner membrane and converted to phosphatidylglycerol and
cardiolipin in the mitochondrial membrane. The junctions return phosphatidylethanolamine, the major outer-membrane lipid, to the
ER for recycling. A similar plastid-associated membrane (PLAM) complex transfers mostly PC between the ER and chloroplasts
and returns digalactosyl-diacylglycerol (DGDG) to the ER for recycling. Some vesicular routes are complemented with nonvesicular
transport, as is the case for ceramide transport between the ER and trans Golgi membranes. The CERT complex (ceramide transfer
protein) connects the Golgi and ER directly. In Gram-negative bacteria, the outer-membrane lipids are derived from the plasma
membrane and are transported by protein complexes across the periplasmic space. Lipopolysaccharide (LPS) transfer complexes
(Lpt) move LPS from the plasma membrane to the outer leaflet of the outer membrane. The outer membrane contains a substantial
number of lipoproteins that anchor the outer membrane to the peptidoglycan layer. Here, Braun’s lipoprotein or murein lipoprotein, a
lipid-anchored short protein (58 amino acids in the mature protein; gene name lpp; PDB accession 1EQ7), is transferred with its lipid
moiety anchored to the Lol shuttle complex from the inner membrane to the inner leaflet of the outer membrane.
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Chapter 9: Metabolism: Growing Membranes, ­Sustaining Cells
and transported from there to the outer membrane via the Lpt transport
protein complex (see Figure 9.19B). This transport complex contains seven
known protein subunits (LptA–G) spanning from the cytoplasmic side of the
inner membrane to the outer leaflet of the outer membrane. Gram-negative
bacteria also have high levels of lipoproteins, small lipid-anchored outermembrane proteins that attach this membrane to the peptidoglycan layer.
Lipoproteins are synthesized and linked to a phospholipid anchor in the
inner membrane and then shuttled to the outer membrane by the Lol transport system. This system is composed of the LolCDE complex, an ABC transporter of the inner membrane, a periplasmic carrier protein LolA, and an
outer-membrane receptor protein LolB. Lol proteins bind the lipoprotein by
its phospholipid anchor, which is subsequently incorporated into the periplasmic leaflet of the outer membrane.
Which side of a membrane a lipid or a lipid-anchored protein ends up is
of course not only important in bacteria but in eukaryotic cells as well. In
fact, not only do all membranes show a distinctly unique lipid composition,
but they also maintain a highly conserved and functionally relevant lipid
asymmetry, as we first discussed in Chapter 2. This asymmetry is actively
maintained by various lipid transporters (flippases) that are either ABC
transporters or P-type ATPases (Figure 9.20). Lipid asymmetry affects signaling, recruitment of peripheral membrane proteins, and shape changes of
membranes (for example, endocytosis, tubulation, and so on). Lipid biosynthesis in eukaryotic cells starts in the smooth ER, where the building
blocks—fatty acids, backbones, head groups (pre-made in the cytoplasm)—
are combined to phosphatidic acid, ceramide, and isoprenes (dolichol).
Initial assembly always starts at the cytosolic surface/side of the ER membrane. Phosphatidic acid is the common precursor for glycerophospholipid
and glyceroglycolipid synthesis, as well as for triacylglycerols (fats and oils).
Sphingolipids like gangliosides and sphingomyelin are made from the common precursor ceramide. Cholesterol and related sterol compounds are
synthesized from isoprene-pyrophosphate precursors. Transmembrane
proteins subsequently shuffle lipids across the bilayer, distributing different
lipids in different ways between the two leaflets of a membrane.
The glycerophospholipids phosphatidic acid, phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine, phosphatidylinositol, and
the related glycosylphosphatidylinositol (GPI) anchor, as well as ceramide
and glucose–ceramide, are incorporated and synthesized on the cytosolic
face of the ER membrane (and, in analogy, on the cytoplasmic side of bacterial cell membranes). To ensure even growth of the lipid bilayer, the ER
flippase—a nonspecific, non-energy-consuming, bidirectional lipid transporter—redistributes these lipids across both ER leaflets and the ER membrane becomes more symmetric with respect to its phospholipid content.
Glycolipid synthesis, although starting at the cytosolic side, proceeds on the
luminal side after the first monosaccharide has been added to the glycerol
or ceramide lipid precursor (for example, forming glucosylceramide). Thus,
a strict lipid asymmetry for oligosaccharides, both for glycolipids and glycoproteins, will be established. This asymmetry is maintained throughout the
trafficking to the cell surface, where small transport vesicles bud off and fuse
with target membranes. This means that the cytoplasmic leaflet components
will always be oriented toward the cytoplasmic compartment, while the
luminal side will always be facing the inside of organellar compartments. If
the plasma membrane is the target, the luminal leaflet fuses with the extracellular leaflet and the luminal content merges with the extracellular fluid. In
fact, the luminal compartments of organelles, while physically separate, are
functionally contiguous with each other and the extracellular side of the cell.
Since phospholipids are distributed symmetrically in the ER membrane,
and the ER is the feeder membrane for the nuclear–endosomal and plasma
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Building Membranes, Building Cells
cholesterol
APLT
PS
ABCA1
PAF,
glycolipids
ABCB1
PC
ABCB4
ER
ABCC1
sterols
ABCG2
Ca2+
floppases (in-out)
ABC transporter
PS, PE
Man, GlcNac2-PP-dol
Man-P-dol
Glc-P-dol
Drs2
GOLGI
GlcN-PI
PL
PC, SM
Drs2
Rft1
nucleus
353
GlcCer
GalCer
GlcCer
GalCer
?
PS, PE
Neo1
Dnf3
PS
PL
Neo1
PL
PL
biogenic flippases
(uni- or bidirectional)
PC, PE, PS
Drs2
Dnf3
Dnf1, Dnf2
scramblases
(bidirectional)
flippases (out-in)
P4-ATPase
Figure 9.20 Regulation of the transbilayer lipid distribution in nuclear–endosomal membrane systems. Biosynthesis of
membrane lipids always starts on the cytoplasmic leaflet of genetic membranes (endoplasmic reticulum [ER], prokaryotic plasma
membrane). To provide proper lipid packing and distribution in both leaflets of a bilayer, and to make and maintain leaflet lipid
asymmetry, lipid-transfer proteins are found in all membranes. The ER and Golgi membranes contain several biogenic flippases;
that is, lipid transporters responsible for the transfer of lipid precursor molecules from the cytoplasmic side to the luminal side
for further head-group synthesis and modification. Bidirectional biogenic flippases found in the ER membrane create leaflet
symmetry for glycerophospholipids (PL) as well as for glucose-ceramide and galactose-ceramide. The Golgi can further modify
sphingoglycolipids on the inner leaflet, but it lacks a flippase to move them to the cytoplasmic surface. It does, however, maintain
symmetry for glucosylceramide (GlcCer) and galactoceramide (GalCer), but creates asymmetry for phosphatidylethanolamine (PE)
and phosphatidylserine (PS) using a flippase. Lipid-anchored oligosaccharides like dolichol-glycolipids and phosphatidylinositolglucosamine for protein glycosylphosphatidylinositol-anchoring are found only on the ER lumen side (for example, Glc-P-dol; glycosylphospho-dolichol). The plasma membrane contains different types of lipid-transfer proteins commonly known as flippases, floppases,
and scramblases. Flippases are P4-type ATPases, always transfer lipids from the lumen/extracellular side to the cytoplasmic side
of a membrane, and are found mostly in the Golgi and transport vesicle membranes and plasma membrane. Floppases are ABCtype transporters and always shuffle lipids from the cytoplasmic to the extracellular side of the plasma membrane, their usual site
of action. Scramblase is a calcium-dependent bidirectional lipid-transfer protein and likely differs from the ER-resident flippases.
The names of lipid substrates are listed in the figure on the receiving leaflet side; names of transporters are on the opposite side
(for example, cholesterol outside and ABCA1 cytoplasmic side). PL, phospholipid; PA, phosphatidic acid; PS, phosphatidylserine;
PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; SM, sphingomyelin; PAF, platelet activating factor;
Cer, ceramide; Glc, glucose; Gal, galactose; Man, mannose; GlcN, glucosamine; GlcNac, N-acetylglucosamine; dol, dolichol;
APLT, aminophospholipid transporter. (Adapted from Daleke DL [2007] J Biol Chem 282:821–825. With permission from ASBMB.)
membranes, which typically show lipid asymmetry, these latter membranes
must have special, unidirectional transport proteins to achieve their phospholipid asymmetry. Indeed, these target membranes have energy-­powered
lipid-translocating enzymes (flippases and floppases) that establish local
asymmetry as needed. Plasma membrane flippases (which are different
from the ER-resident biogenic flippases) are P-type ATPases that move
amino-phospholipids (PE and PS) from the extracellular to the cytoplasmic
leaflet (out → in); floppases are ABC transporters that move phospholipids,
cholesterol and ceramides, and sphingomyelin from the cytoplasmic to the
extracellular (or luminal) side (in → out); and scramblases ‘deteriorate’ the
asymmetry by moving lipids randomly and independent of energy across
the bilayer. This passive process leads to an equilibration of lipid composition in both monolayers.
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Chapter 9: Metabolism: Growing Membranes, ­Sustaining Cells
Proteins have special signal sequences to find their
destination membrane
Figure 9.21 Insertion mechanisms
for membrane proteins. Membrane
protein insertion in eukaryotes can be
co- or post-translational. Co-translational
insertion (1) is a signal-recognition particle
(SRP)/ribosome-dependent transport across
the translocon (Sec61), with the membranespanning segments sequentially released
laterally into the lipid bilayer. The only site
of co-translational insertion is the rough
endoplasmic reticulum (ER) membrane.
Several post-translational mechanisms
are found in the following situations. Tailanchored proteins (2) are inserted via
a single membrane-spanning segment
close to the C-terminal end of the protein.
In this case, the protein is released from
cytoplasmic ribosomes and held stable by
chaperones until the C-terminal segment
can be recognized by the Get1–3 insert
machinery. Lipid-anchored proteins (3)
are usually released fully folded into the
cytoplasm and post-translationally attached
to a lipid anchor (myristic and palmitic
acids, glycosylphosphatidylinositol [GPI]
anchor, or farnesyl and geranyl prenol
anchors). The lipid type specifies the target
membrane and location. Acyl and prenyl
anchors link proteins to the cytoplasmic
side of a membrane. GPI-anchored
proteins are linked to the anchor in the ER
lumen and trafficked via vesicle transport
to the extracellular side of the plasma
membrane. Finally, mitochondrial and
plastid proteins (4) are post-translationally
inserted into the outer membranes of their
respective organelles by protein import
complexes (TOM in mitochondria and TOC
in chloroplasts). A small percentage of
proteins can be inserted post-translationally
into ER membranes by Sec62/63
complexes. This type of insertion requires
chaperone proteins to keep the nascent
polypeptide in an unfolded conformation for
the Sec, as also required for TOM and TOC
translocation complexes.
CellMembranes ch09.indd 354
Membranes of course contain lots of proteins, and membrane protein
synthesis, too, starts at special cellular locations followed by trafficking
and sorting of proteins to the proper cellular (or extracellular) location. In
eukaryotes, proteins of the nuclear–endosomal membrane system, including the plasma membrane, are inserted into the rough ER and distributed
from there via the Golgi complex to the proper target membrane. Membrane
insertion depends on the translocon linked to synthesis on the ribosomes,
a mechanism known as co-translation. Proteins of the mitochondria and
plastids (and in some cases peroxisomes) and nuclear membrane are
inserted post-translationally. Interestingly, protein export and insertion of
transmembrane proteins involves the same ER-resident machinery—the
translocon (resident in plasma membranes in prokaryotes) (Figure 9.21).
Translocons are found in the rough ER membrane (ribosomes are docked
to translocons) and are responsible for the import of newly synthesized proteins into the ER lumen. Membrane proteins are directly embedded into the
ER membrane by the same translocon complex. The newly emerging polypeptide chains wind through the translocon pore, from where the protein’s
hydrophobic (membrane-spanning) segments are directly released laterally
into the lipid bilayer, where the protein folds and protein subunits assemble
into functional membrane protein complexes (see also Figure 7.31). Thus,
in eukaryotes, secretion and membrane insertion of proteins start with a
proxy compartment, the ER lumen, followed by membrane trafficking using
elaborate vesicle-based sorting and transport mechanisms along the secretory pathway. Trafficking moves exocytic vesicles and their luminal cargo to
the cell surface. When the vesicle membranes fuse with the plasma membrane, they release their luminal cargo into the extracellular space through
fusion pores. The bilayers of the vesicles may carry membrane proteins,
which as a result of fusion are inserted into the plasma membrane, since the
entire vesicle membrane and all of its components become a part of the cell
­surface. The topology of the membrane is strictly enforced, meaning that the
cytoplasmic side of the vesicle membrane faces the cytoplasmic side in the
plasma membrane, while the luminal side faces the extracellular side.
Some proteins are not inserted into membranes in a co-translational fashion, but are guided to membranes only after translation is finished and the
nascent protein is released from the ribosome. Post-translational insertion
starts with the protein being synthesized on cytosolic ribosomes and released
into the cytoplasm prior to binding to the translocon, or an alternative
1 ribosome/SRP, Sec61
2 tail-anchored (TA) Get1–3
chaperone
ribosome
plasma membrane
ER
N
nucleus
C
C
4
2,4
3
N
2,3,4
mitochondrion
2
1,4
3 lipid-anchoring
acyl, prenyl, GPI
N
N
chloroplast
4 chaperones (Hsp70),
Sec62/63, TOC, TOM
C
chaperones
N
N
C
C
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355
integrase in the ER for tail-anchored proteins. Mitochondrial and plastid
proteins rely on protein import complexes (TOM, TOC) in the respective
organelles. Translocons, integrases, and import complexes shuttle proteins
across membranes (or integrate them into the bilayer) in an unfolded conformation. To prevent premature folding, nascent proteins are recognized
by chaperones and guidance proteins that prevent them from aggregating or
folding by keeping them soluble in the aqueous environment of the cytosol.
The proteins are then guided to their target membranes of mitochondria,
chloroplasts, and peroxisomes. Proteins targeted for the nucleus are translocated co-translationally to the ER membrane and from there move laterally
to the outer nuclear membrane. To reach the inner nuclear membrane, they
pass along the membrane tunnels of the nuclear pore complexes.
For co- and post-translational insertion/translocation, special N- or
C-terminal signal sequences are used to put the proteins into or across the
proper target membrane (Table 9.4). Co-translational insertion into the
ER membrane (plasma membrane in prokaryotes) requires an N-terminal
signal sequence recognized by the signal-recognition particle (SRP). Posttranslational signals vary depending on the targeted organelle. For mitochondria and chloroplasts, an N-terminal sequence is used; for peroxisomes
and nuclear proteins (not membrane proteins, but for the nuclear lumen), a
C-terminal signal sequence determines translocation.
Once inserted into the target membrane, many proteins need to be transported to a different membrane in the cell. The signals for this sorting or
trafficking process vary. For cell surface and extracellular proteins, a glycosylation unit serves as the proper sorting signal. Added in the ER lumen, it
is part of a proofreading mechanism to only allow properly folded proteins
to be released away from the ER; the particular carbohydrate content of the
glycosylated unit can be modified on the way to the target location.
Finally, lipid-anchored proteins (via acylation, prenylation, or GPI anchoring) are inserted post-translationally. The type of lipid anchor determines
the membrane location of the protein. GPI-anchored proteins are found on
the extracellular side of the plasma membrane. Accordingly, GPI anchors are
attached in the lumen of the ER. The protein is first synthesized as a membrane protein with a C-terminal, membrane-embedded signal sequence.
The extramembranous, luminal N-terminal domain is then cleaved from the
transmembrane anchor and moved onto the pre-made GPI lipid anchor.
In contrast, the common myristoylation of lipid-anchored proteins occurs in
the cytoplasm, where the C14 fatty acid is covalently linked to an N-terminal
glycine residue. A short sequence of positively charged residues close to the
modified N-terminus (see Figure 6.17), together with the fatty acid chain,
promotes the surface anchoring of these proteins to lipid domains rich in
phosphatidylinositol (for example, PIP2).
Table 9.4 Signal sequences to target membrane proteins to the proper target membrane
Target membrane
Sequence location
Sequence
Translocon
Cell membrane
N-terminal or internal
MMSFVSLLLVGILFYATEAEQLTKCEVFQ
SecY
ER (cell membrane)
N-terminal or internal
MMSFVSLLLVGILFYATEAEQLTKCEVFQ
Sec61
Glycosylation
AXN
Get3p
C-terminal
(S/A/C)-(K/R/H)-(L/A), most common SKL
Post-translational
N-terminal
(R/K)-(L/V/I)-XXXXX-(H/Q)-(L/A/F)
Mitochondrion
N-terminal
MLSLRQSIRFFKPATRTLCSSRYLL
TOM/TIM
Chloroplast
N-terminal
MAMAMRSTFAARVGAKPAVRGARPASRMSCMA
TOC/TIC
Peroxisome
CellMembranes ch09.indd 355
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Chapter 9: Metabolism: Growing Membranes, ­Sustaining Cells
Fusion and fission are central to maintaining
and building cells
Cells and their cell membranes originate from parent cells and membranes. To make more cells, these membranes have to be grown, divided,
and reassembled. Thus, membrane fusion and fission are central to building
and maintaining cells. Membrane fusion events are important for plasma
membrane repair and maintenance. This includes fusion events along the
secretory pathway, from fusion of ER-derived transport vesicles with the cis
Golgi membrane and controlled exocytosis—that is, vesicle fusion with the
plasma membrane (Figure 9.22). Many organelles undergo regulated fusion
events including where small vacuoles merge to form a larger vacuole, multiple short mitochondria fuse into long tubular reticula, and endosomes
fuse with each other or with lysosomes. Additional fusion events include
cell fusion during sexual reproduction (yeast mating types, sperm and egg
cells) and, finally, viral entry such as by human immunodeficiency virus
(HIV) and influenza viruses that have a membrane-derived viral envelope
(for additional details, see Chapter 7). A larger variety of fusogenic proteins
is involved in mediating contact and bilayer fusion between corresponding membranes, which guides localization and proper target membrane
identification.
Figure 9.22 Various fusion events in
eukaryotic cells. Membrane fusion events
are important in growing and maintaining
cellular membranes. Shown here for
animal and yeast cells, fusion events
are involved in the secretory pathway
(endoplasmic reticulum [ER] to Golgi to
plasma membrane) leading to exocytosis
and plasma membrane repair, in the fusion
of organelles including endosomes and
lysosomes, vacuoles (also in plants), and
mitochondria (chloroplasts in plants), in
cell fusion, and in viral entry sites, either
on the plasma membrane (for example,
human immunodeficiency virus) or within
lysosomes (for example, influenza). Various
fusion proteins are involved to ensure
the proper control and location of fusion
events; the SNARE complexes are the most
ubiquitous. Ig, immunoglobulin. Preferred
fusion proteins at various sites are as
follows: plasma membrane repair–SNARE/
dysferlin/myoferlin/tricalbin; regulated
exocytosis–SNARE/synaptotagmin;
vacuol fusion–SNARE/HOPS; ER-Golgi
and endosome/lysosome fusion–SNARE/
tethering factors; mitochondrial fusion–
OPA1/Mgm1/Mitofusin/Fzo1; cell-cell
fusion–Ig-domain proteins/actin bundles.
(Adapted from Martens S & McMahon HT
[2008] Nat Rev Mol Cell Biol 9:543–556.
With permission from Macmillan Publishers
Ltd.)
CellMembranes ch09.indd 356
For membrane maintenance, where old membrane pieces are recycled and
replaced with newly synthesized components, fusion activity is matched
with corresponding fission events to avoid growth or shrinkage of the cell
membrane area (Figure 9.23). Membrane fission causes fragmentation of
a larger membrane into two smaller ones. One of the most important separations is cell division. Cell division requires not only the fission of plasma
membranes, but also growth of the nuclear endomembrane system and
the replication of mitochondria and chloroplasts. The proliferation of mitochondria and plastids, as well as the fragmentation and reconstitution of
the endomembrane systems in higher eukaryotes during mitosis, are particularly notable processes. Each type of organelle has unique issues when
it comes to fusion and fission. Mitochondria, chloroplasts, and the nucleus
have double-membrane envelopes and thus have to find ways of synchronously processing both membranes and maintaining their topological
ANIMAL CELL
YEAST CELL
viral entry
plasma
membrane
repair
cell–cell
fusion
site
regulated
exocytosis
Golgi
ER
endosome–
lysosome
fusion
cell–cell
fusion site
endosome
fusion
mitochondrial
fusion site
SNAREs
dysferlin, myoferlin, tricalbin
viral fusion protein
OPA1/Mgm1
synaptotagmin
vacuole fusion
tethering/regulatory factors
Ig-domain proteins
mitofusin/Fzo1
HOPS
actin bundles
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Building Membranes, Building Cells
(A)
PLANT CELL
ANIMAL CELL
CCV
phagosome
(B)
caveolae
viral budding
ESCRT
Golgi
Vps4
vacuole
lysosome
ER
nucleus
plasma
membrane
chloroplast
cell
plate
actin
comet
vacuole
mitochondrion
mitochondrial
fusion site
cleavage
furrow
dynamine ring
outside neck
Vps4
ESCRT
early
endosome
ER
nucleus
Golgi
ESCRT
Vps4
midbody
viral
ribonucleoprotein
dynamin-like protein
plant dynamin
OPA1/Mgm1
Mx
cytoplasm
phagocytosed bacterium
cytoplasm
lumen/
extracellular
(C)
MVB
endosome
peroxisome
classical dynamin
mitofusin/Fzo1
357
ESCRT-III ring
inside neck
lumen/
extracellular
Figure 9.23 Various fission events in eukaryotic cells. (A) Organelle remodeling and membrane bending leading to fission,
using dynamin or dynamin-like GTPases for fission of membranes released into the cytosolic compartment of the cell. Dynaminbased fission is found in endocytosis, organelle fission, and endosomal membrane trafficking. Dynamin is also involved in tubulation
mechanisms related to fusion of mitochondria and cell-plate-forming vesicles in plants. In this case, the membrane is elongated rather
than separated. ER, endoplasmic reticulum; CCV, clathrin-coated vesicle. (B) ESCRT-III (‘endosomal sorting complex required for
transport’) scission functions in late endosome formation of luminal multivesicular bodies (MVBs), in human immunodeficiency virus
budding from the cell surface, and in animal cytokinesis. Vps4 is an AAA-ATPase and is responsible for the removal of ESCRT proteins
after fission is completed. (C) Topology of membrane fission or scission differs between dynamin GTPase rings that constrict the neck
from the outside, and ESCRT-III scission machinery, that attaches and separates the membranes from the inside of the fission neck
structure. Dynamin-mediated fission releases the vesicle toward the cytoplasmic compartment; ESCRT fission releases the vesicle into
the lumen or the extracellular compartment (outside) of the cell. (A, adapted from Praefcke GJ & McMahon HT [2004] Nat Rev Mol
Cell Biol 5:133–147. With permission from Macmillan Publishers Ltd.; B, adapted from Hill CP & Babst M [2012] Biochim Biophys Acta
1823:172–181. With permission from Elsevier Inc.)
arrangement in the process—that is, that the outer and inner membranes
(and thylakoid membranes in chloroplasts) are not mixed up during either
fusion or fission and retain their relative order or topology (outer membranes remain outer membranes, and so on).
The importance of topology—that is, how budding membranes retain their
relative location regarding the outside, cytosolic compartment, and luminal
side—is also evident in the direction of membrane fission; that is, whether
budding releases membrane vesicles toward the cytoplasmic or the luminal/
extracellular side of the membrane. To discriminate between the two types
of fission, budding is mediated by two different types of fission proteins: the
dynamin and dynamin-related GTPases that form a constrictive ring around
the outside of the membrane neck; and ESCRT proteins, which form a constrictive ring on the inside of the fission neck.
CellMembranes ch09.indd 357
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Chapter 9: Metabolism: Growing Membranes, ­Sustaining Cells
Dynamin-controlled fission is used for releasing membranes into the cytoplasmic compartment. Dynamin-mediated fission events are by far the
most common type and include all forms of endocytosis, the budding of
secretory vesicles along the secretory pathway from the ER and Golgi membranes, as well as the fission of peroxisomes, mitochondria, and chloroplasts. Interestingly, dynamin activity does not always result in fission; it is
also used for tubulation processes as found in the elongation of the cell plate
(formed from fused vesicles) during plant cell division and in membrane
tubulation at the mitochondrial fusion site.
ESCRT proteins release the membrane pieces into the lumen of the organelle—specifically, the endosomal compartment—and out of the cell during
viral release (for example, influenza or HIV budding) and cell division. Cell
division mechanisms come in various forms that are characteristic for a type
of cell or taxonomic group. Three basic types of cell division mechanisms
that depend on membrane fission include binary fission in prokaryotes,
budding in single-cell yeast, and cytokinesis in animal cells. Cell fission usually leads to a symmetric split of a growing cell into two equal daughter cells.
Fission includes the growth of a cleavage furrow in animal cells, which is
guided by a central fission ring that grows from the surface to the cell center
and ends in the complete separation of the constricted cell membrane.
(A)
hypha
growth tips
septa with pores
(B)
cell wall
plasma
membrane
ER
nucleus
Golgi vesicles
nucleus
future location of
plasmodesmata
ER
CellMembranes ch09.indd 358
Yet the ultimate outcome of a cell division is not simply the formation of
newly formed, individual cells. Rather, it is a process often integrated into
tissue formation, be these filamentous microorganisms or the organs of
multicellular organisms. In filamentous microorganisms, the cell division
need not result in complete fission; instead, dividing cells do not separate
and grow septa containing cytoplasmic bridges between them. These septa
maintain the filamentous cell organization and communication in fungi,
algae, and bacteria (Figure 9.24). Here, a fungal hypha grows an undivided,
multinucleated cytoplasmic compartment. This single long compartment is
subsequently partitioned by septa that contain large central pores that allow
cytoplasmic streaming and exchange of entire organelles. Bacteria undergo
symmetric binary fission but can also use septa formation during filamentous life-cycle stages and sporulation. Single yeast cells can divide by asymmetric budding or symmetric fission of a larger cell into equal daughter cells.
This latter division is reminiscent of animal cell cytokinesis, where cells first
grow and then split symmetrically, forming a cleavage furrow. In contrast,
plant cells grow a fused cell plate from internal Golgi-derived vesicles. Cellplate formation grows around ER tubules, forming the future sites of primary plasmodesmata, the membrane tunnels connecting the cytoplasms of
neighboring plant cells.
There are many other situations that require special mechanisms to deal
with membrane growth and cell maintenance, such as the fragmentation
and reassembly of the nuclear envelope during mitosis. During mitosis in
Figure 9.24 Cellular compartmentation in plants and filamentous fungi. (A) In
many organisms, cell division events do not end in fission but septum formation,
leaving the growing organisms in a filamentous stage, as shown here for a
branching fungal hypha. The growth cone of a hypha elongates forming a long,
multinucleated cytoplasm. Cell partition is achieved later by septum formation. For
this, the septum grows inward from the cell membrane and cell wall in a process
similar to formation of the cleavage furrow in animal cells. However, the process is
not completed and leaves a septum with a single large pore allowing cytoplasmic
exchange of even large organelles. (B) Plant cells divide from within, growing small,
Golgi-derived vesicles with luminal cell wall components that fuse along the central
plate and eventually form two fully functional but attached cells. Endoplasmic
reticulum (ER) tubules reaching across the emerging cell plate from both newly
separated nuclei prevent complete cell-plate fusion and contribute to the formation
of primary plasmodesmata.
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Building Membranes, Building Cells
(A)
ER
(B)
NPC
NUCLEUS
INM
metaphase
late anaphase
interphase
telophase
ONM
ER LUMEN
CYTOSOL
GFP
LBR
NUCLEOPLASM
endogenous
LBR
eukaryotic cells, the duplicated chromosomes are attached to the microtubule spindle apparatus. In most eukaryotes—with the exception of diatoms,
dinoflagellates, and some single-celled yeast—the attachment of microtubules to the chromosomal kinetochore complexes is preceded by the fragmentation of the nuclear envelope, whose membranes become part of the
flexible, tubular ER network (Figure 9.25). Nuclear inner-membrane proteins that interact with chromosomes, such as the lamin B receptor, freely
diffuse within the nuclear/ER membrane network after fragmentation; they
can then guide the nuclear envelope reconstitution during late anaphase
and telophase, where the endosomal membranes embedded with lamin B
receptors bind to the clustering chromosomes.
Finally, membrane reorganization contributes to the formation of more
complex tissue structure, such as the development of vascular tubes from
the fusion of intracellular vacuoles with intercellular plasma membranes
in animals (for formation of blood vessels, and so on) (Figure 9.26). Large
numbers of pinocytic vesicles undergo fusion inside the cell to form an elongated vacuole-like compartment that will eventually fuse on each side of the
endothelial cell. Vacuoles from neighboring cells essentially fuse together to
then form a new, extracellular compartment.
359
Figure 9.25 Model of nuclear envelope
reassembly. (A) The nuclear and
endoplasmic reticulum (ER) membranes
form a contiguous membrane system (top
panel). Inner nuclear membrane (INM)
proteins such as the lamin B receptor
(LBR) that bind chromatin fibers to the
inner nuclear membrane can move laterally
from their site of synthesis in the rough
ER to their site of activity (bottom panel).
(In the study shown, the localization of
LBR was studied by fusion with green
fluorescent protein, GFP.) ONM, outer
nuclear membrane. (B) In most eukaryotic
cells, mitosis requires the fragmentation
of the nuclear envelope prior to the
interaction of the spindle apparatus with
the chromosomes. The nuclear membranes
become part of the ER network. Inner
nuclear membrane proteins like LBR diffuse
throughout the ER network (light-purple
circles). After separation of duplicated
chromosomes and dissociation of the
microtubule spindle apparatus, the
growing and stretched-out ER membranes
reassemble a nuclear envelope around
each set of chromosomes. The key to
nuclear membrane reassembly around
separated, condensed chromosomes is
the LBR proteins (dark-purple circles) that
can attach to chromosomes freed from
the spindle apparatus during telophase.
(Adapted from Ellenberg J, Siggia
ED, Moreira JE et al. [1997] J Cell Biol
138:1193–1206. With permission from The
Rockefeller University Press.)
This process is reminiscent of plasmodesmata formation in plants during cellplate formation in cytokinesis. This demonstrates that similar mechanisms
(A)
(B)
cytoplasm
cytoplasm
nucleus
vacuole
lumen
CellMembranes ch09.indd 359
Figure 9.26 A model for vascular
lumen formation by intracellular and
intercellular fusion of endothelial
vacuoles. Vascular tubes can form between
endothelial cells that first undergo internal
vacuole formation. Vacuoles are likely
derived from endocytosis and grow larger
by fusion inside each cell (intracellular
fusion); this is followed by vacuole–cell
membrane fusion, forming a central
vascular tube (intercellular fusion). The
model is shown (A) in side view and (B) as
the view along the lumen tube. (A, adapted
from Kamei M, Saunders WB, Bayless KJ
et al. [2006] Nature 442:453–456. With
permission from Macmillan Publishers Ltd.;
B, Mostov K & Martin-Belmonte F [2006]
Nature 442:363–364. With permission from
Macmillan Publishers Ltd.)
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Chapter 9: Metabolism: Growing Membranes, ­Sustaining Cells
are used in seemingly very different physiological circumstances, but have
similar outcomes—the formation of large compartments in multicellular
organisms that could not possibly be achieved by growing extra-large single
cells. Differences in outcome are of course important. While the vacuolar
fusion in animal tissues builds a true extracellular compartment (the vascular tube), vesicle fusion from cell-plate formation creates true intracellular
connectors through cytoplasmic bridges, potentially creating a large supercellular cytoplasm in plant tissues.
Chapter Summary
Cell membranes are an integral component of metabolism. They enclose
metabolically and genetically distinct compartments, have their own
enzymatic machinery, use transporters to connect and regulate metabolic
pathways, and extract and convert environmental energy for cellular use.
Membrane bioenergetics deals with cell membrane transport processes that
control the formation and dissipation of ion gradients and the formation of
high-energy compounds like ATP and NADPH. Ion gradients store energy
in the form of an electrochemical potential. This energy can be converted
into other forms of energy including chemical, transport, and mechanical
energy. The electrochemical potential is available to organisms for biosynthesis (photosynthesis and respiration), transport of metabolites (absorption and secretion), mechanical work (bacterial flagellar rotor, swimming,
crawling), and signaling processes (action potentials). As lipid synthesis and
energy metabolism are membrane-bound processes, cell membranes can
be rightfully considered as metabolic compartments in the same way that
aqueous compartments like the cytoplasm or mitochondrial matrix are considered metabolic compartments. Membranes are self-renewing structures
that grow and divide by fusion and fission processes. However, cells cannot
synthesize membranes from scratch and, once lost, membranes cannot be
regenerated. Thus all modern membranes are derived from ancient membranes and in present-day cells come in independently renewing membrane
lineages—bacterial plasma membranes and the eukaryotic nuclear–endomembrane system, as well as mitochondrial and chloroplast membranes.
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