INTRACELLULAR COMPARTMENTS AND PROTEIN SORTING
1. Describe the organization of the eucaryotic cell and know the functions of the major
organelles.
2. Understand the importance of sorting signals for directing a protein to a particular
location within the cell.
3. Describe the structure of the nuclear pore complex. Describe how proteins are
transported between the nucleus and the cytosol
4. Describe the major biochemical reactions that occur in peroxisomes. Describe how
proteins are imported into peroxisomes.
5. Understand the steps required for protein targeting to the endoplasmic reticulum.
6. Describe how different topological arrangements of transmembrane proteins are
achieved.
7. Understand the role of dolichol in tranferring oligosaccharides to proteins in the
endoplasmic reticulum.
8. Know the compartments that communicate by vesicular transport. Describe the
formation of clathrin-coated vesicles.
9. Understand the role of the Golgi apparatus in protein sorting. Know the locations in
the cell that require the Golgi apparatus for protein targeting.
10. Describe the process of protein recycling from the Golgi apparatus to the endoplasmic
reticulum.
11. Understand the role of oligosaccharide attachement to targeting of lysosomal
hydrolases.
12. Describe the role of lysosomes in cellular digestive processes. Describe how defects
in lysosomal hydrolases can lead to disease.
13. Distinguish between constitutive and regulated secretion.
14. Describe the different pathways for delivering endocytosed or cellular materials to
lysosomes for digestion.
15. Describe the process of receptor-mediated endocytosis using LDL as an example.
Understand how mutation to the LDL receptor causes familial hypercholesterolemia.
16. Describe how early endosomes function as sorting stations for endocytosed material.
Describe the possible fates of the receptor during receptor-mediated endocytosis.
INTRACELLULAR COMPARTMENTS AND PROTEIN SORTING
1. Organization of eucaryotic cell into membrane-enclosed organelles
a. Nucleus- contains chromosomal DNA; site of DNA and RNA synthesis
b. Cytoplasm- everything outside of nucleus (cytosol and cytoplasmic organelles)
 Endoplasmic reticulum (ER)
- Rough ER- Site of synthesis of proteins that are destined for secretion or
for certain other organelles; studded with ribosomes; proteins synthesized
on ER begin synthesis in cytosol and are translocated during synthesis
- Smooth ER- Section of ER that does not contain ribosomes; lipid
synthesis; detoxification (in liver); Ca2+ storage (especially in
sarcoplasmic reticulum of muscle)
 Golgi apparatus- Receives proteins and lipids from ER and sends to
appropriate destination; often covalently modifies them
 Mitochondria- generate most of cell’s ATP
 Lysosomes- contain digestive enzymes that degrade unwanted organelles and
material received from outside by endocytosis
 Endosomes- used for passage of endocytosed material on its way to lysosomes
 Peroxisomes- contain enzymes for various oxidative reactions
 Cytosol- space in cytoplasm outside of organelles; protein synthesis and
degradation; intermediary metabolism
2. Transporting proteins to organelles
a. Synthesis of all proteins begins in cytosol; proteins can be directed to various
locations
b. Mechanisms of transporting proteins
 Gated transport- Continuity between nucleus and cytosol exists by nuclear
pore complexes that perforate nucleus; provide selective gate for transporting
certain proteins
 Transmembrane transport- Proteins interact with membrane-bound protein
translocators; translocators contain aqueous pore through which protein is fed;
protein usually must unfold to go through translocator
 Vesicular transport- Vesicles pinch off from membrane of one compartment,
thereby containing proteins from that compartment; fuse to membrane of
another compartment and discharge cargo
c. Sorting signals
 Segment(s) of amino acids within a protein serve as signals to direct them to a
particular organelle
 Types of sorting signals
- Signal sequence- continuous stretch of amino acids; many located at one
end and removed by signal peptidases after sorting completed
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Signal patch- three-dimensional arrangement of amino acid on surface;
residues can be distant to each other in primary sequence
Sorting signals recognized by sorting receptors that guide protein to
appropriate compartment
Most proteins have no sorting signal and remain in cytosol
Variability in signal sequences on different proteins that are targeted to same
organelle; physical properties of amino acids (hydrophobicity, charge, etc.)
often more important than exact sequence
Experiments to determine functional signal sequence in protein directed to a
particular organelle; if segment functions as a signal sequence, adding it to a
normally cytosolic protein will result in that protein being targeted to the
organelle
3. Transport between nucleus and cytosol
a. Occurs through nuclear pore complexes that perforate nuclear membrane; large
multi-protein complex composed of proteins called nucleoporins
b. Aqueous channels in nuclear pore complex allow small molecules to pass by
diffusion; most proteins too large to diffuse through nuclear pore complex; gating
mechanism allows only selected proteins to pass
c. Proteins transported from cytosol to nucleus contain nuclear localization signal
(NLS); NLS's can exist as signal sequences or signal patches; can be located
almost anywhere in protein; often rich is positively charged amino acids (arginine
and lysine)
d. Nuclear import receptors; family of related receptors that each recognizes a group
of proteins containing similar NLS's; associate with NLS-containing cargo protein
in cytosol
e. Cargo-bound nuclear import receptors receptor bind nucleoporins at many sites
which mediates transport through nuclear pore complex; receptor disassociates
from cargo in nucleus and recycled back to cytosol
f. Nuclear export occurs by similar process operating in reverse; nuclear export
receptors bind nuclear export signals on cargo proteins; cargo-bound receptors
bind nucleoporins
g. Proteins remain folded while passing through nuclear pore complex
4. Transport into peroxisomes
a. Functions of peroxisomes
 Site of enzymes that oxidize organic substrates to produce hydrogen peroxide
(H202) (RH2 + O2 → R + H202)
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Contains catalase that utilizes H202 to oxidize other substrates (H202 + R'H2 →
R' + 2H20) or converts H202 to H20 (2 H202 → 2H20 + O2); detoxify molecules
especially in liver and kidneys
Oxidative reactions break down fatty acid molecules
Site of initial reactions in synthesis of plasmalogens, abundant phospholipid in
myelin sheaths that insulate nerve cell axons; many peroxisomal disorders
lead to neurological disease
b. Import from cytosol
 Signal sequence often located at C-terminus; some proteins have a different
signal sequence near N-terminus
 Proteins known as peroxins participate in transport process; includes soluble
receptors in cytosol that recognize signal sequences and docking proteins on
membrane of peroxisome
 Proteins do not have to unfold during import
 Zellweger syndrome- group of inherited defects in essential peroxins such as a
membrane protein involved in import; empty peroxisomes; severe
abnormalities in brain, liver and kidneys, die soon after birth
 Milder disorder from defective receptor for the N-terminal import signal
5. Transport into mitochondria
a. Mitochondria have own genome that encodes some of their proteins;
mitochondrial genome maternally inherited
b. Most mitochondrial proteins encoded by nuclear genome; these proteins
synthesized in cytosol, imported using sorting signals and sorting receptors
6. Transport into endoplasmic reticulum
a. ER is site of synthesis for all proteins destined for secretion, the plasma
membrane, lysosomes, endosomes, the Golgi, or the ER itself
b. Docking protein onto ER membrane
 Signal sequence contains string of hydrophobic amino acids
 Signal recognition particle (SRP) binds to signal sequence as soon as it
emerges from ribosome; pauses translation
 Protein is transported to ER co-translationally; complex containing protein on
ribosome and SRP binds to SRP receptor in ER membrane and is brought to
translocator; SRP and receptor disassociate
 Signal sequence also acts as start transfer sequence to open translocator;
translation continues as protein is fed through translocator
c. Soluble protein into ER lumen
 Signal sequence at N-terminus
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Protein transported to ER membrane co-translationally following synthesis of
signal sequence
Translation continues as translocation of protein through ER membrane
begins
Cleavage site adjacent to signal sequence recognized by signal peptidase;
protein released into ER lumen
d. Single-pass transmembrane protein containing N-terminal signal sequence
 Co-translational docking to ER and translocation through membrane begins as
for soluble protein containing N-terminal signal sequence
 Additional internal hydrophobic segment binds to translocator and acts as
stop-transfer sequence; causes release of protein from translocator
 Stop-transfer sequence remains as membrane-spanning segment
e. Single-pass transmembrane protein containing internal signal sequence
 Protein recognized by SRP and brought to ER following synthesis of internal
signal sequence; one segment of protein fed through translocator
 Signal sequence remains as membrane-spanning segment
 Two orientations of signal sequence determine which end of protein is
inserted into ER lumen
f. Multi-pass transmembrane protein- multiple internal start and stop tranfer
sequences result in multiple membrane spanning segments
7. Glycosylation of proteins synthesized in ER
a. Most proteins made in ER are glycoproteins
b. N-linked glycosylation- oligosaccharide precursor added to NH2 group on side
chain of certain asparagine residues in ER; extensive processing in Golgi
subsequently removes some sugar residues from N-linked oligosaccharides
c. Preformed oligosaccharide precursor composed of 14 residues transferred to
protein; transfer occurs in ER during translation as soon as asparagine to be
gycoslylated enters lumen; catalyzed by oligosaccharyl transferase
d. Oligosaccharide precursor to be transferred attached to a lipid called dolichol in
ER membrane; attachment by high-energy pyrophosphate bond that drives
transfer reaction
e. Synthesis of dolichol-linked oligosaccharide precursor
 Stepwise addition of sugar resides
 Begins on cytosolic face with formation of nucleotide-sugar intermediates that
donate sugars to dolichol
 Dolichol flipped across membrane partway through process using flippase in
ER membrane
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Reactions on lumenal face involve sugar transfer from monosaccharide-linked
dolichol molecules that are themselves synthesized on cytosolic face from
nucleotide-sugar intermediates and subsequently flipped
f. Incompletely processed proteins retained in ER
g. O-linked glycosylation- oligosaccharide linked to hydroxyl group on side chains
of serine, threonine, or hydroxylysine residues; less frequent; occurs in Golgi
8. Protein folding in ER
a. Chaperones in ER help prevent aggregation of unfolded proteins
b. Improperly folded proteins transported from ER to cytosol through reverse action
of translocator; deglycosylated, ubquitylated, and degraded by proteasome
9. Addition of glycosylphosphatidylinositol (GPI) anchor in ER
a. Occurs on some proteins destined for plasma membrane
b. Protein embedded in ER membrane by hydrophobic C-terminal sequence
c. Enzyme cuts protein free from C-terminal sequence and attaches preassembled
GPI; signal contained in C-terminus and few adjacent amino acids
10. Mechanisms of vesicular transport
a. Vesicle buds off from one compartment and fuses with another; carries cargo
from lumen and membrane of donor compartment to target compartment;
compartments that communicate by vesicular transport are topologically
equivalent
b. Protein coats in vesicular transport
 Coated vesicle formed from cage of proteins covering cytosolic surface
 Coating concentrates membrane proteins that are transported and deforms
patch of membrane to mold forming vesicle; coat removed before fusing with
target
 Different types of coated vesicles used for different transport steps
c. Clathrin-coated vesicles
 Used for transport vesicles from Golgi and plasma membrane
 Clathrin subunit composed of three large and three small chains called
triskelion; triskelions assemble into convex framework of hexagons and
pentagons on cytosolic surface of membranes; introduce curvature leading to
formation of bud
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Clathin is linked to transmembrane cargo receptors by adaptins; different
types of adaptins for different cargo receptors
Dynamin is GTPase that assembles around neck of bud and aids in pinching
off; pinching off involves membrane fusion at neck
Clathrin coat removed after transport vesicle is pinched off
11. Transport from ER through Golgi apparatus
a. Organization of Golgi apparatus
 Golgi stack is series of flattened membrane-bound compartments known as
cisternae; divided into cis, medial, and trans cisternae
 Adjacent to cis cisterna is cis-Golgi network (CGN) and adjacent to trans
cisterna is trans-Golgi network (TGN); CGN and TGN each composed of
interconnected tubular and cisternal structures
 Proteins pass from ER to CGN, through cis, medial and trans cisternae, and
then to TGN; proteins in TGN are sorted and transported to other
compartments or to cell surface
 Processing of oligosaccharide chains occurs in the different parts of the Golgi
apparatus
b. Transporting proteins from ER to CGN
 Transported using coated vesicles
 Selective process involving ER exit signal increases efficiency
 Proteins without exit signals still exit ER but less efficiently
c. ER resident proteins
 Proteins that reside in ER have sorting signals known as ER retrieval signals
that bring them back to ER if they enter the Golgi apparatus
 ER resident proteins have retrieval signals at C-terminus, KKXX for
membrane proteins and KDEL (lysine-aspartic acid-glutamic acid-leucine) for
soluble proteins
 Retrieved membrane proteins interact directly with coat proteins on retrograde
transport vesicles
 Retrieved soluble proteins bind to transmembrane KDEL receptor, which
interacts with coat proteins
d. Processing of N-linked oligosaccharides; formation of two main classes of Nlinked oligosaccharide that share common core containing two Nacetylglucosamines (GlcNAc) and three mannoses
 Complex oligosaccharides
- Core region plus terminal region containing variable number of copies of
GlcNAc-galactose-sialic acid unit
- Formed by trimming oligosaccharide precursor made in ER to core and
then sequentially adding sugar residues that are donated by sugarnucleotide intermediates
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High-mannose oligosaccharides- precursor made in ER not trimmed all the
way to core so most mannose residues remain
12. Transport from TGN to lysosomes
a. Function of lysosomes- contain acid hydrolases for controlled digestion of
macromolecules; pH maintained at 5.0 for maximal activity of enzymes
b. Sorting lysosomal hydrolases by recognition of attached M6P
 Mannose 6-phosphate (M6P) specifically added to N-linked oligosaccharides
of lysosomal hydrolases in CGN
 In TGN M6P-containing hydrolases bind to transmembrane M6P receptors,
which interact with coat proteins; coated vesicles bud off and are delivered to
late endosomes, which mature into lysosomes
c. Addition of M6P to hydrolases- signal patch recognized by GlcNAc
phosphotransferase; adds GlcNAc-phosphate to terminal mannose residue;
GlcNAc cleaved off by another enzyme so that M6P exposed
d. Lysosomal storage diseases
 Genetic defects affecting lysosomal hydrolases; accumulation of undigested
material in lysosomes; most severe pathological consequences often in
nervous system
 Most disorders are defects in a gene for a lysosomal hydrolase
- Tay-Sachs disease has defect in the glycosidase hexosaminidase A; results
in accumulation of ganglioside GM2, which is present in plasma
membranes particularly in nerve cells
- Gaucher disease has defect in glucocerebrosidase; results in accumulation
of glucocerebroside
- Hurler’s disease has defect in -L-iduronase that breaks down
glycosaminoglycans
 I-cell disease- most hydrolases missing from lysosomes; inclusion bodies
containing undigested material form in lysosomes; defect in GlcNAc
phosphotransferase gene so that hydrolases are not targeted to lysosomes but
are secreted
13. Transport to cell surface (exocytosis); proteins sorted for at least three pathways in
TGN- lysosomes and two secretory pathways
a. Constitutive secretory pathway
 Transport vesicles bud from TGN and fuse with plasma membrane
 Supplies plasma membrane with lipids and transmembrane proteins; soluble
proteins are secreted to extracellular space
 Known as default pathway because proteins do not require specific signal
after they reach ER
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b. Regulated secretory pathway- occurs in specialized secretory cells; sorting signal
targets proteins to special secretary vesicles; remained stored until extracellular
signal stimulates secretion
c. Proteins being secreted or going to lysosomes are all initially directed to ER by
signal sequence that is recognized by SRP
14. Transport into cell from surface (endocytosis)
a. Uptake of macromolecules by cells; material to be ingested becomes enclosed by
plasma membrane as it invaginates; buds off to form endocytic vesicles
b. Endocytic/degradation pathways- how endocytosed or cellular materials can
travel to lysosomes for digestion
 Endocytosis
- Pinocytosis (cell drinking)- fluid and solutes continually ingested in small
vesicles; often involves formation of coated pits
- Receptor-mediated endocytosis- internalize specific macromolecules from
extracellular fluid; macromolecules bind specific transmembrane
receptors, which interact with clathrin protein coat; form coated pits that
bud off into coated vesicles
- Phagocytosis- uptake of large particles such as microorganisms or dead
cells; generally performed by specialized cells in immune system
 Autophagy- obsolete organelles enclosed by membranes to form
autophagosome which fuses with lysosome
c. Uptake of cholesterol by cells- example of receptor-mediated endocytosis
 Cholesterol transported in blood as particles known as low-density
lipoproteins (LDL); LDL particles contain many molecules cholesterol
esterified to fatty acids; organized by single protein that mediates binding of
LDL particle to transmembrane LDL receptor
 LDL receptor interacts with clathrin-coated pit
 Individuals with LDL receptor mutation that causes defective binding site for
coated pit have increased risk of heat attack from atherosclerosis (familial
hypercholesterolemia)
d. Early endosome
 Endocytoic vesicles fuse with early endosomes
 Act as sorting station of endocytosed material
 Acidic environment often causes ligand and receptor to dissociate; ligand
usually digested in lysosomes
 Possible fates of receptor
- Recycling- Receptor returned by transport vesicles to plasma membrane
- Transcytosis- in polarized cell receptor can also be transported specifically
to other domain of membrane
- Degradation- receptors not retrieved are degraded in lysosomes
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For receptor-mediated endocytosis of LDL, receptor is recycled back to
plasma membrane while LDL is degraded in lysosomes to release free
cholesterol
e. Pathway from early to late endosomes and to lysosomes
 Early endosome migrates toward cell interior
 Form multivesicular bodies by enclosing their own invaginated membrane,
which makes membrane proteins fully accessible for digestion
 Turn into late endosomes either by fusing with preexisting ones or with each
other; late endosomes more acidic
 Late endosomes converted to lysosomes by receiving hydrolases from TGN
and further acidification
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