Essential
Cell Biology
Third Edition
Chapter 15
Intracellular Compartments and
Transport
Hilary Truchan
truchanhk@vcu.edu
Copyright © Garland Science 2010
What we see in most textbooks:
and what it really looks like:
Figure 15-1 Essential Cell Biology (© Garland Science 2010)
The real numbers
Table 15-2 Essential Cell Biology (© Garland Science 2010)
Overview
• Briefly describe membrane-enclosed organelles of
eukaryotic cells and their functions
• Discuss how the protein composition of each
organelle/compartment is formed and maintained
• Discuss how organelles communicate with each
other
• Vesicles
Membrane-Enclosed Organelles
Internal Membranes Create Enclosed Compartments
and Organelles - Segregating Metabolic Processes
Examples:
- Separate
glycolysis from
glycogenesis
- Separate
synthesis of
protein bonds
from hydrolysis of
protein bonds
Figure 15-1 Essential Cell Biology (© Garland Science 2010)
Basic set of Organelles Found in Most Animal Cells
Figure 15-2 Essential Cell Biology (© Garland Science 2010)
Functions of Organelles
Table 15-1 Essential Cell Biology (© Garland Science 2010)
Evolutionmembranes
of the ER and may
Nuclear
Membranes
Intracellular
have
evolved from
invagination of the plasma membrane
Single compartment
Plasma membrane invaginated forming a twolayered envelope of membrane surrounding the
DNA
Endomembrane System
Figure 15-3 Essential Cell Biology (© Garland Science 2010)
Protein Sorting
How are proteins sorted into discrete locations?
• Cell must duplicate its membrane-enclosed
organelles before dividing
• Most organelles formed from preexisting
organelles then divide and are distributed
between daughter cells
• Non-dividing cells continuously generate proteins
and replace proteins that have been degraded
• Proteins need to be sorted correctly
– organelle membrane proteins, organelle lumen
proteins, secreted proteins
– HOW? 3 Mechanisms
How can proteins cross a phospholipid bilayer?
1. Nuclear Pores
1. Cytosol into the Nucleus
– Nuclear pores - penetrate
the inner and outer
membranes
– Selective gates
– Transport specific molecules
– Allow passive diffusion of
smaller molecules
Figure 15-5 Essential Cell Biology (© Garland Science 2010)
How can proteins cross a phospholipid bilayer?
2. Protein Translocators
2. Cytosol into ER,
mitochondria, chloroplast
– Transported across
membrane by protein
translocators - located in the
membrane
– Protein usually has to unfold
to snake through the
membrane
– Similar to bacteria
Figure 15-5 Essential Cell Biology (© Garland Science 2010)
How can proteins cross a phospholipid bilayer?
3. Transport Vesicles
3. ER onward and from one
compartment of
endomembrane system to
another
– Transport vesicles - loaded
with cargo of proteins from
the interior space of one
compartment
– Discharge cargo into second
compartment by fusing with
the membrane
– Membrane components also
delivered (lipids and proteins)
Figure 15-5 Essential Cell Biology (© Garland Science 2010)
Signal Sequences
- direct proteins
to correct organelle
Localization
sequences
Conserved AA sequence that acts as a molecular “address”
telling the cell where this protein needs to live in the cell!
Table 15-3 Essential Cell Biology (© Garland Science 2010)
If localization signals are removed, the protein does
not arrive at the required destination
Figure 15-6 Essential Cell Biology (© Garland Science 2010)
How do Proteins Enter the Nucleus?
Architecture of the nucleus
Nuclear envelope - defines nuclear
compartment - formed from two concentric
membranes
Inner nuclear membrane - contain proteins
that act as binding sites for the
chromosomes and provide anchorage for
the nuclear lamina
Nuclear lamina - protein filaments that
provide structural support for the
nuclear envelope
Outer nuclear membrane - membrane
similar composition as the ER
membrane (continuous with)
Nuclear pores - form the gates which
all molecules enter or leave the
nucleus
Figure 15-7 Essential Cell Biology (© Garland Science 2010)
Nuclear Pore Complex – A Gate
Nuclear pores contain ~30 different proteins
H2O filled passages - small water-soluble molecules can pass freely between nucleus
and cytosol
Jumble meshwork of proteins inhibit larger molecules from passing through the pore
Nuclear Localization Sequence (NLS)
Larger molecules need an NLS to pass through the pore - 1 or 2 short
sequences of positively charged lysines or arginines
Figure 15-8a Essential Cell Biology (© Garland Science 2010)
EM of Nuclear Pores
EM - side view of two nuclear pore complexes
EM - face-on view of nuclear pore complexes
Figure 15-8b Essential Cell Biology (© Garland Science 2010)
Nuclear transport receptors actively transport
proteins through nuclear pores
NTR - grab onto sequences within the tangle of
nuclear pore to carry the cargo into the nucleus
Figure 15-9 Essential Cell Biology (© Garland Science 2010)
Importing Proteins into the Nucleus Requires Energy
- GTP hydrolysis
Similar process
used to carry
mRNA out of the
nucleus in the
cytoplasm
Proteins remain in fully
folded conformation!
Different than transport
mechanisms into other
parts of the cell.
Figure 15-10 Essential Cell Biology (© Garland Science 2010)
How do Proteins Enter the Mitochondria and
Chloroplasts?
© Sarah E Golding PhD.
Proteins unfold in order to enter Mitochondria and
Chloroplasts
HELPED BY CHAPERONES TO TRANSFER
AND RE-FOLD!
Proteins that enter have an N-terminal signal sequence (red)
Proteins translocate across both membranes simultaneously at specific sites
Proteins are unfolded as they are transported
Signal sequence removed after translocation completed
Figure 15-11 Essential Cell Biology (© Garland Science 2010)
How do Proteins Enter the Endoplasmic Reticulum?
ER - most extensive membrane system in a
eukaryotic cell
• Entry point for proteins destined for other organelles as
well as the ER itself
• Proteins destined for the Golgi apparatus, lysosomes,
endosomes, cell surface all first enter the ER from the
cytosol
• Once in ER proteins do
not return to the
cytosol but rather
travel via vesicles
Figure 15-12 Essential Cell Biology (© Garland Science 2010)
© Sarah E Golding PhD.
2 kinds of proteins transferred from the cytosol to the
ER
1. Water-soluble proteins - translocated across
ER membrane into the ER lumen
•
Destined for secretion or for the lumen of an
organelle
2. Prospective transmembrane proteins partially translocated across ER membrane
and become embedded in it
•
•
Destined to stay in ER membrane, membrane of
another organelle, or plasma membrane
Directed to ER by ER signal sequence - 8 or
more hydrophobic amino acids
Proteins that enter
the ER begin to
enter the ER
membrane before
the polypeptide
chain has been
completely
synthesized
Figure 15-13 Essential Cell Biology (© Garland Science 2010)
ER localization signals are recognized by SRPs
ER signal sequence guided to the ER membrane by:
1. A signal-recognition particle (SRP) - in the cytosol binds to the ER signal
sequence
when it is exposed on the ribosome
2. An SRP receptor - embedded in membrane of the ER - recognizes the SRP
Figure 15-14 Essential Cell Biology (© Garland Science 2010)
Soluble proteins cross ER to enter lumen
• ER signal sequence - usually N-terminus - functions to
open channel
• Remains bound to channel as remainder of protein
chain threaded through membrane as a large loop
• ER signal cleaved once proteins have crossed!
Figure 15-15 Essential Cell Biology (© Garland Science 2010)
How are Transmembrane Proteins Transported into
the Membrane?
Single-pass Transmembrane Proteins
ER signal cleaved once proteins have crossed!
Figure 15-16 Essential Cell Biology (© Garland Science 2010)
Double-pass Transmembrane Protein
Start transfer-sequence - internal signal sequence
used to start the protein transfer - never removed from peptide!
Figure 15-17 Essential Cell Biology (© Garland Science 2010)
Multi-pass Transmembrane Proteins
Need additional pairs of stop and start
sequences
– One sequence reinitiates translocation further down
peptide chain
– The other stops translocation and causes
polypeptide release and so on
– Stitched into membrane as they are synthesized
Vesicular Transport
http://www.youtube.com/watch?v=rvfvRgk0MfA
Vesicular Transport
• Two types – secretory pathway and endocytic
pathway
• Secretory pathway
– Entry into the ER is the first step - pathway to another
destination
– Initial destination is the Golgi apparatus
– From Golgi to other compartments - carried out by
budding and fusion of transport vesicles
– Extend outward: ER  plasma membrane
• Endocytic pathway
– Extend inward: plasma membrane  lysosomes
– COMMUNICATION between cells!
Secretory and Endocytic Pathways
Secretory pathway - RED arrows
Endocytic pathway - GREEN arrows
Figure 15-18 Essential Cell Biology (© Garland Science 2010)
Vesicle Budding – Assembly of a Protein Coat
•
•
•
•
Vesicles that bud from membranes have a
distinctive protein coat on cytosolic surface coated vesicles
After budding from parent organelle - sheds the
coat allowing the vesicle to interact directly with
the membrane it will fuse to
Cells produce different types of coated
vesicles – focus on Clathrin
Two functions of the coat:
1. Shapes the membrane into a bud
2. Helps capture molecules for onward transport
Clathrin coated vesicles
http://www.youtube.com/
watch?v=eRslV6lrVxY
Figure 15-19b Essential Cell Biology (© Garland Science 2010)
Clathrin forms a “cage” to carry vesicles to the
membrane
• Clathrin - best studied vesicles have coats made largely of clathrin
• Bud from the Golgi apparatus on the outward secretory pathway
• Bud from the plasma membrane on the inward endocytic pathway
•Electron micrograph (EM) showing a clathrin-coated vesicle forming
•Assemble into a basketlike network on the cytosolic surface of the membrane
•starts shaping the membrane into a vesicle
Figure 15-19a Essential Cell Biology (© Garland Science 2010)
Clathrin coated vesicles transport select cargo
molecules
Adaptins - secure clathrin coat to vesicle membrane and help select the cargo molecules
Dynamin - small GTP binding protein - assembles around neck of invaginated coated pit
Causes ring to contrict - pinching off vesicle from membrane
Hydrolysis of GTP and help of other
proteins to pinch off vesicle
Figure 15-20 Essential Cell Biology (© Garland Science 2010)
Adaptin proteins are specific to destination
Different adaptins - adaptins that bind cargo receptors in the plasma membrane
not the same as those that bind cargo receptors in the Golgi apparatus
-Reflects differences in cargo molecules from each source
Table 15-4 Essential Cell Biology (© Garland Science 2010)
How are vesicles transported through the cytosol?
• Actively transported by motor proteins that
move along the cytoskeleton
• Learn More about this in Chapter 17
http://www.biozentrum.unibas.c
h/research/groupsplatforms/overview/unit/schoen
enberger/
Vesicle Reaches Target - Recognize and Dock with
the Organelle
Rab proteins - surface of the vesicle are recognized by tethering
proteins on the cytosolic surface of the target membrane
Specific combination of Rab proteins and tethering proteins - identify membrane
type
Ensure vesicles fuse only with the correct membrane
SNAREs - transmembrane proteins important for
docking the vesicle in place
SNAREs on the vesicle (v-SNAREs) interact with complementary
SNAREs on the target membrane (t-SNAREs)
Vesicle Fusion - deliver its cargo and adds
vesicle membrane to organelle
Figure 15-21 Essential Cell Biology (© Garland Science 2010)
Membrane fusion sometimes needs a specific
molecular signal
•
Fusion requires the two lipid bilayers come within 1.5nm of each other so lipid can
intermix
• Need to displace water from hydrophilic surface of the membrane
• After pairing the v-SNAREs and t-SNAREs wrap around each other = pulls two
membranes into close proximity
Figure 15-22 Essential Cell Biology (© Garland Science 2010)
Intracellular bacteria
• Chlamydia spp.
• Mycobacterium
• Salmonella
• WHY? Reduced genome sizes compared to extracellular
bacteria
• E. coli = 4.6 megabases
• Chlamydia = 1.3 megabases
• Do not have the genes to synthesize many
essential nutrients – e.g. Amino acids
• Must parasitize these from their host!
• Take advantage of vesicular trafficking and
hijack nutrient-rich vesicles!
Many intracellular bacteria target Rab Proteins!
• Mycobacterium tuberculosis acquires iron and other
nutrients by targeting recycling endosome and transGolgi Rab Proteins
• Chlamydia spp. acquire sphingolipids and amino acids
by targeting trans-Golgi Rab Proteins
http://www.rki.de/EN/Content/Institute/Departments
Units/JuniorGroups/JRG5.html
Uninfected human epithelium cells (left) with compact Golgi band close to the
cell nucleus (blue) and cells infected with Chlamydiae trachomatis with Golgi
fragments (red) which accumulate around the bacterial inclusion (green).
How do proteins cross the plasma membrane?
Secretory Pathways
Exocytosis
• Exocytosis - newly made proteins, lipids, and
carbohydrates are delivered from the ER, via
the Golgi apparatus, to the cell surface by
transport vesicles that fuse with the plasma
membrane
• FIXED sequence of membrane-enclosed
compartments - often chemically modified en
route
Chemical modifications which occur in the ER
• Disulfide bonds are formed between cysteine
side chains
Remember? Stabilize protein structure
• Glycosylation - covalent attachment of short
oligosaccharide side chains
Remember? Protect extracellular proteins, serve
as transport signal form carbohydrate layer, help
with cell to cell recognition
– Carried out by glycosylating enzymes in ER but
not in the cytosol
Glycosylation
• Glycosylation - addition of oligosaccharide side chains at asparagine residue
residues added en bloc
• 14-sugar oligosaccharide is originally attached to specialized lipid in ER
membrane – dolichol
• Not all proteins in ER are glycosylated. Requires specific tripeptide sequenc
adjacent to the modified asparagine.
This is the beginning of
protein modification!
Proteins are further
modified in the Golgi.
Figure 15-23 Essential Cell Biology (© Garland Science 2010)
Regulated ER Exit – protein quality control!
Proteins that function in ER - have ER retention signal
Exit from ER highly selective
Proteins that fold incorrectly or multi-meric proteins that fail to assemble properly
are retained in ER and bind to chaperone proteins
Chaperone proteins holds proteins in ER until proper folding occurs - if does not
occur proteins are degraded
Figure 15-24 Essential Cell Biology (© Garland Science 2010)
Unfolded protein response (UPR)
Unfolded protein response (UPR)
- when cells protein production
exceeds carrying and folding
capacity
UPR prompts the cell to make
more ER
- including all molecular
machinery (lots of
transcription!)
UPR allows cell to adjust size of
ER to met the cellular needs
- if misfolded proteins continue to
accumulate (out of control!)
- UPR can direct cell to undergo
apoptosis
Figure 15-25 Essential Cell Biology (© Garland Science 2010)
Proteins further modified and sorted in the Golgi
apparatus
• Oligosaccharides added in ER are further
modified
– removing or adding sugars
Golgi apparatus
• Usually located near cell nucleus
• Collection of flattened membrane-enclosed sacs
called cisternae with two distinct faces:
– Cis face is adjacent to the ER
– Trans face points toward the plasma membrane
• Outermost cisterna on each face is connected to
a network of tubules and transport vesicles
The Golgi apparatus! Molecular post office
Proteins enter at Cis-face: move through and exit Golgi
or return to ER
Proteins exit at trans-face: transported to Plasma
membrane or lysosomes
Figure 15-26 Essential Cell Biology (© Garland Science 2010)
Secretory proteins are released by exocytosis
Figure 15-18 Essential Cell Biology (© Garland Science 2010)
Secretory cells - Regulated and Constitutive (operates continually) Pathways
Constitutive Secretion vs Regulated Secretion
Constitutive secretion – all Eukaryotic cells  NO Signal Sequence
• Proteins incorporated into plasma membrane, extracellular matrix or are signaling
molecules
Regulated secretion – specialized cells  need signal to stimulate fusion with the
plasma membrane and release cargo to cell exterior
•
Example: Insulin!
Figure 15-27 Essential Cell Biology (© Garland Science 2010)
Insulin is released by regulated secretion
Example: Release of insulin from a secretory vesicle of a pancreatic  cell
Signaled to release by an increase in glucose levels in the blood
Figure 15-28 Essential Cell Biology (© Garland Science 2010)
How do proteins enter the cell? Endocytic Pathways
Review video: http://www.youtube.com/watch?v=SGBiy1HlWH8
Endocytosis
• Endocytosis - eukaryotic cells continuously take
up fluid, as well as large and small molecules
• Material to be ingested enclosed by a small
portion of plasma membrane - buds inward and
then pinches off to form an intracellular
endocytic vesicle
2 main types of Endocytosis
• Pinocytosis (“cellular drinking”) - ingestion of
fluid and molecules via small vesicles (<
150nm in diameter)
– Carried out predominantly by clathrin-coated
vesicles
• Phagocytosis (“cellular eating”) - ingestion of
large particles via large vesicles called
phagosomes (generally > 250nm in diameter)
– Only “phagocytic” cells do this
Phagocytic cells can ingest whole cells!
Figure 15-32 Essential Cell Biology (© Garland Science 2010)
Macrophage engulfing a
pair of red blood cells
Receptor-mediated Endocytosis
Pinocytosis is indiscriminate
Receptor-mediated Endocytosis = controlled
pinocytosis
– More efficient - macromolecules bind to complementary
receptors on the cell surface and enter the cell as
receptor-macromolecule complexes in clathrin-coated
vesicles
– Selective concentrating mechanism
– Increases efficiency of internalization of particular
macromolecules more than 1000-fold compared to
pinocytosis
– Example: Cholesterol
LDL enters cells by receptor-mediated endocytosis
Figure 15-33 Essential Cell Biology (© Garland Science 2010)
Endosomes – site for sorting of imported
macromolecules
• Two sets of endosomes
– Early endosomes: Just beneath plasma membrane mature into late endosomes as they fuse with each
other
– Late endosomes: Closer to the nucleus
• Interior of endosome kept acidic by proton pump
in the endosomal membrane
– pH 5-6
– Low pH causes receptors to release their cargo
• Main sorting station in the inward endocytic
pathway
– trans-Golgi is the sorting station for exocytic or
secretory pathway!
Routes taken by
receptor once they
enter the endosome
differ according to the
receptor type
1. Recycling
2. Lysosomes
3. Transcytosis
Figure 15-34 Essential Cell Biology (© Garland Science 2010)
Lysosomes are the Principal Sites of Intracellular
Digestion
Lysosome contains
hydrolytic enzymes and a
proton pump
Digest worn out organelles
and extracellular materials
pH ~5 (acidic)
Unique membrane contains transporters to
allow products of the
digestion to be transferred to
the cytosol to be either
excreted from cell or used
by the cell
Figure 15-35 Essential Cell Biology (© Garland Science 2010)
Acidic pH and
destructive
enzymes are
contained within
membrane!
The lysosome - The cell dumping station!
Endocytosed,
phagocytosed and
autophagosomal
material are trafficked to
the lysosome for
destruction!
Autophagy is the
destruction of an
organelle – envelopes
organelle and takes it to
the lysosome
Figure 15-36 Essential Cell Biology (© Garland Science 2010)