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Alberts • Johnson • Lewis • Raff • Roberts • Walter
Molecular Biology of the Cell
Fifth Edition
Chapter 13
Intracellular Vesicular Traffic
Copyright © Garland Science 2008
The cell continually adjusts the composition of its plasma membrane
Figure 13-1 Molecular Biology of the Cell (© Garland Science 2008)
Transport vesicles bud from one membrane and fuse with another,
carrying a cargo (soluble molecules in it)
Figure 13-2 Molecular Biology of the Cell (© Garland Science 2008)
The “road-map” of the biosynthetic secretory and endocytic pathway
Figure 13-3 Molecular Biology of the Cell (© Garland Science 2008)
Figure 13-3b Molecular Biology of the Cell (© Garland Science 2008)
Vesicular transport
• Mediates continuous exchange of components between chemically
distinct membrane-enclosed compartments.
• molecular markers displayed on the cytosolic surface of the membrane
serve as guidance for traffic, to ensure that vesicles fuse only with the
correct compartment.
• A specific combination of markers gives each compartment its
molecular address
• Cells segregate (accumulate in one space) proteins into separate
membrane domains by assembling a special protein coat on the
membrane’s cytosolic surface.
Coated vesicles
• Coats are needed for budding
• Before fusion happen the coats are removed from the vesicles
• The function of the coats is to:
- Concentrate specific membrane proteins in a specific patch,
which then makes a vesicle
- Coat molds (shapes) the forming vesicle
Coat proteins assemble into a basket like curved lattice, and so
shapes the vesicle.
• Same types of coats make similar shaped / sized vesicles
Three different types of coats cover transport vesicles
Figure 13-4 Molecular Biology of the Cell (© Garland Science 2008)
Each coat is used for a different transport step
Figure 13-5 Molecular Biology of the Cell (© Garland Science 2008)
Clathrin coated pits
Figure 13-6 Molecular Biology of the Cell (© Garland Science 2008)
Clathrin protein is the major component f clathrin-coated vesicles
Composed of 3 large and three small polypeptides, together form the
triskelion
Figure 13-7a, b Molecular Biology of the Cell (© Garland Science 2008)
The clathrin triskelion determines the geometry of the clathrin cage
Figure 13-7c, d Molecular Biology of the Cell (© Garland Science 2008)
Adaptor proteins are a major component in clathrin-coated vesicles, they
form a second layer of the coat, they capture cargo receptors
The sequencial assembly of adaptor complexes and clathrin on the
cytosolic side of the membrane generate forces that results in the
formation of a clathrin-coated vesicle
Figure 13-8 Molecular Biology of the Cell (© Garland Science 2008)
Not all coats form basket-like structures: retromer assemble on
endosomes to Golgi
Figure 13-9 Molecular Biology of the Cell (© Garland Science 2008)
RETROMER ASSEMBLY
Retromer assembles when:
1. It can bind to the cytoplasmic side of the cargo receptors
2. It can interact directly with a curved phospholipid bilayer
3. It can bind to a specific phospholipid: phosphorylated
phosphatydilinositol, which act as an endosomal maker.
PHOSPHOINOSITIDES MARK ORGANELLES AND MEMBRANE
DOMAINS
• Phosphoinositides can undergo rapid cycles of phosphorylation and
dephosphorylation at te 3’, 4’ and 5’ end to produce various PIPs.
• Different organelles in the endocytic and secretory pathway have
distinct sets of PI and PIP Kinases and phosphatases.
Figure 13-10 Molecular Biology of the Cell (© Garland Science 2008)
PIP-BINDING PROTEINS HELP REGULATE VESICLE
FORMATION AND OTHER STEPS IN MEMBRANE TRANSPORT
Figure 13-11 Molecular Biology of the Cell (© Garland Science 2008)
Cytoplasmic proteins regulate the pinching-off and un-coating of coated
vesicles
• Dynamin assemble as a ring around the neck of each bud, using GTP energy it works
to help seal off the forming vesicle. Dynamin binds to PI(4,5)P2 which tethers the
protein on the membrane.
• After vesicle forms, clathrin falls off with the help of an Hsp70 ATPase; a PIP
phosphatase that is in the vesicles depletes PI(4,5)P2
Figure 13-12a Molecular Biology of the Cell (© Garland Science 2008)
Cytoplasmic proteins regulate the pinching-off and un-coating of coated
vesicles- role of dynamin
Figure 13-12b Molecular Biology of the Cell (© Garland Science 2008)
Monomeric GTPases control coat assembly
To balance vesicular traffic to / from compartment, coat proteins
must assemble only when and where they are needed:
• PIPs play a major role in regulating the assembly of clathrin coats
on the PM and golgi, there are in addion:
• Coat-recruitment monomeric GTPases: control the assembly on
endosomes and the COPI and COPII coats on golgi and ER:
• These include Arf proteins (COPI and clathrin at golgi)
• Sar1 (COPII on ER)
Usually these are found in the cytosol at high concentrations, in
an inactive GDP-state.
Monomeric GTPases control coat assembly: Sar1 activation
Figure 13-13a Molecular Biology of the Cell (© Garland Science 2008)
Active Sar1 GTP now recruits coat (COPII) protein subunits to initiate
budding
Figure 13-13b Molecular Biology of the Cell (© Garland Science 2008)
COPII protein components
Figure 13-13d Molecular Biology of the Cell (© Garland Science 2008)
Sec13/31 assemble into a cage
Figure 13-13c Molecular Biology of the Cell (© Garland Science 2008)
NOT ALL TRANSPORT VESICLES ARE SPHERICAL
• Clathrin coats have to produce considerable force to bend the plasma
membrane, especially at the neck of the bud where Dynamin is
required for pinching of he vesicle.
• Budding occurs preferentially at regions where the membrane are
already curved, such as the rims of the Golgi cisternae.
• Vesicles are dynamic and of different shapes and sizes: long tubules
leave the trans Golgi network, vesicle pinch off from the tubules.
RAB PROTEINS GUIDE VESICLE TARGETING
•Rabs are monomeric GTPases: they can function on transport
vesicles, on target membranes or both.
• They cycle between the cytoplasm and the membrane (regulated
by GAPs and GTPases).
• Rab GDI (Rab-GDP dissociation inhibitor) binds to GDP-bound
state to keep them soluble inactive in the cytoplasm.
• In their GTP state they bind to effectors called: Rab effectors that
facilitate vesicle transport, membrane tethering and fusion.
RAB PROTEINS GUIDE VESICLE TARGETING
Table 13-1 Molecular Biology of the Cell (© Garland Science 2008)
RAB PROTEINS ROLE IN TETHERING DOCKING AND FUSION
Figure 13-14 Molecular Biology of the Cell (© Garland Science 2008)
SNAREs MEDIATE MEMBRANE FUSION
Figure 13-16 Molecular Biology of the Cell (© Garland Science 2008)
SNAREs MEDIATE MEMBRANE FUSION
• After tethering, the vesicle fuses to the target membrane:
• Specialized fusion proteins overcome energy barrier to catalyze
membrane fusion:
• SNARE proteins do this and also participate in specificity (35 different
SNAREs)
• SNAREs exist as complementary sets: v-SNAREs (a single protein) on
vesicles and t-SNAREs (two or three proteins) on target membranes.
A model of how SNARE proteins mediate membrane fusion
Figure 13-17 Molecular Biology of the Cell (© Garland Science 2008)
SNAREs must be disassembled before they can function in each
fusion events: NSF ATPase does that
Figure 13-18 Molecular Biology of the Cell (© Garland Science 2008)
Skip Viral fusion paragraph 764-766
Page 766 Molecular Biology of the Cell (© Garland Science 2008)
The recruitment of cargo molecules into ER transport vesicles
(COPII)
ER exit sites: where vesicles bud
Figure 13-20 Molecular Biology of the Cell (© Garland Science 2008)
To exit the ER proteins must be properly folded: chaperones help in
this folding.
Figure 13-21 Molecular Biology of the Cell (© Garland Science 2008)
Homotypic fusion: fusion of membranes from the same
compartment: requires matching SNAREs
Figure 13-22 Molecular Biology of the Cell (© Garland Science 2008)
Structures that form when ER derived vesicles fuse together:
Vesicular-tubular clusters, making a new compartment.
Figure 13-23a Molecular Biology of the Cell (© Garland Science 2008)
Structures that form when ER derived vesicles fuse together:
Vesicular-tubular clusters, making a new compartment; short lived, move along
micotubules.
Retrieval, retrograde transport from the vesicular tubular clusters
happens by COPI vesicles.
Figure 13-23b Molecular Biology of the Cell (© Garland Science 2008)
The retrieval pathway to the ER uses sorting signals: KDEL is a
resident sequence
Figure 13-24a Molecular Biology of the Cell (© Garland Science 2008)
KDEL receptor is needed, it has high affinity to cargo in VTC and
low affinity in the ER
Figure 13-24b Molecular Biology of the Cell (© Garland Science 2008)
The Golgi apparatus consists of an ordered series of compartments:
4-6 cisternae in the Golgi
Figure 13-25a Molecular Biology of the Cell (© Garland Science 2008)
The Golgi is localized near the nucleus, held by microtubules
Figure 13-25b Molecular Biology of the Cell (© Garland Science 2008)
Each Golgi has two distinct faces: cis face (entry) and trans face (exit):
CGN and TGN
Figure 13-25c Molecular Biology of the Cell (© Garland Science 2008)
Figure 13-26a Molecular Biology of the Cell (© Garland Science 2008)
Different enzymes localize to different Golgi areas
Figure 13-27 Molecular Biology of the Cell (© Garland Science 2008)
Different enzymes localize to different Golgi areas
Figure 13-28 Molecular Biology of the Cell (© Garland Science 2008)
The Golgi is prominent in cells that are specialized for secretion: goblet
cells secrete mucus in the intestinal epithelium
Figure 13-29 Molecular Biology of the Cell (© Garland Science 2008)
DIFFERENT ENZYMES LOCALIZE TO DIFFERENT GOLGI
AREAS
Resident Golgi proteins are all membrane bound,
So the ER and the Golgi are organized in different ways.
OLIGOSACCHARIDES ARE PROCESSED IN THE GOLGI
APPARATUS: 2 classes of N-linked oligosaccharides
Figure 13-30 Molecular Biology of the Cell (© Garland Science 2008)
Ordered pathway of sugar modifications in the Golgi
Figure 13-31 Molecular Biology of the Cell (© Garland Science 2008)
N- and O- linked glycosylation
Figure 13-32 Molecular Biology of the Cell (© Garland Science 2008)
DIFFERENT ENZYMES LOCALIZE TO DIFFERENT GOLGI
AREAS
• N-linked glycosylation promotes protein folding:
Makes folding intermediates more soluble, therfore
preventing their aggregation.
the sequential modifications of the N-linked
oligosaccharides establish a code that helps chaperones in binding.
• Makes a protein more resistant to digestion
• Protection against pathogens
• cell – cell recognition : development
• Signaling
Transport through the Golgi may occur by Vesicular transport or by
Cisternal maturation
Figure 13-35 Molecular Biology of the Cell (© Garland Science 2008)
Figure 13-35a Molecular Biology of the Cell (© Garland Science 2008)
Figure 13-35b Molecular Biology of the Cell (© Garland Science 2008)
Page 779 Molecular Biology of the Cell (© Garland Science 2008)
Lysosomes contain soluble hydrolytic enzymes
• Lysosomes membrane are highly
glycosylated, well protected from the
lysosomal proteases
• Enzymes used for digestion of
macromolecules
Figure 13-36 Molecular Biology of the Cell (© Garland Science 2008)
LYSOSOMES ARE HETEROGENEOUS IN SHAPE AND SIZE
Figure 13-37 Molecular Biology of the Cell (© Garland Science 2008)
MODEL FOR LYSOSOME MATURATION
No real distinction between lysosomes and late endosomes, they are
at different stages of the maturation cycle.
Figure 13-38 Molecular Biology of the Cell (© Garland Science 2008)
Skip p781 section “plant…”
MULTIPLE PATHWAYS DELIVER MATERIALS TO LYSOSOMES
From ER golgi  lysosomes
Other pathways also include:
• From endocytosed material  early endosomes  late endosomes
• Autophagy: in liver cells for ex. the mitochondria lives 10 days
Autophagy process is highly regulated and selected cell components
can be marked for lysosomal destruction: form autophagosomes; can
remove large organelles or protein complexes.
MODEL OF AUTOPHAGY
4 steps in autophagy:
1. Nucleation and extension, englufs part of cytoplasm
2. Closure of autophagosome
3. Fusion with lysosomes
4. Digestion of the inner membrane of the autophagosome
Figure 13-41 Molecular Biology of the Cell (© Garland Science 2008)
SUMMARY OF THREE PATHWAYS TO DELIVER MATERIAL TO
LYSOSOMES
Figure 13-42 Molecular Biology of the Cell (© Garland Science 2008)
TRANSPORT OF PROTEINS TO LYSOSOMES
• Proteins are co-translationally
transported into the rough ER, then go to
the Golgi and TGN
• In the TGN proteins carry a marker
mannose 6-phosphate (M6P) groups
added to the N-linked oligosaccharides
• M6P receptors in the TGN recognize
these , cclathrin coat assembles and the
vesicles are delivered to the early
endosomes. M6P receptor binds to M6P at
high pH and release it at low pH.
Figure 13-43 Molecular Biology of the Cell (© Garland Science 2008)
TRANSPORT OF NEWLY SYNTHESIZED LYSOSOMAL
HYDROLASES TO LYSOSOMES
Note: retromer is needed for recycling the M6P receptor to the golgi
Figure 13-44 Molecular Biology of the Cell (© Garland Science 2008)
A signal patch in the hydrolase polypeptide chain provides the cue for
M6P addition
• M6P are added only to the appropriate glycoprotein in the golgi apparatus
• a Signal patch is the signal to add the M6P
• Two enzymes work sequencially: GlcNAc phosphotransferase in cis golgi adds
GlcNAc-phosphate; a second enzyme in the trans golgi cleaves the GlcNAc leaving
behind the M6P marker.
Figure 13-45 Molecular Biology of the Cell (© Garland Science 2008)
DISEASES RELATED TO LYSOSOMES
Genetic defects that affect the lysosomal hydrolases cause lysosomal
storage diseases: results in an accumulation of undigested substrates
in lysosomes , with sever pathological consequences, in nervous
system.
In Inclusion-cell disease, almost all of the hydrolytic enzymes are
missing from the lysosomes of fibroblasts, forming large inclusions,
the hydrolases fail to sort properly so they are secreted to the blood,
because of a defective GlcNAc phosphotransferase.
SOME LYSOSOMES DO EXOCYTOSIS
• In skin cells, the pigment making cells called melancytes store their
piment in lysosome compartments called malanosomes.
• The melanosomes fuse with the membrane to empty the pigments
which are then taken up by kratinocytes, leading to bormal skin
pigmentation.
• In some genetic disorders defects of melanosome exocytosis leads
to hypopigmentation = albinism
Page 787 Molecular Biology of the Cell (© Garland Science 2008)
Phagocytosis : cell uses a large endocytic vesicle: phagosome to ingest
large particles
• Professional phagocytes:
Macrophages and neutrophils,
defend against infections
• the phagosome fuse with
lysosomes inside the cell and
ingested material is degraded.
Figure 13-46 Molecular Biology of the Cell (© Garland Science 2008)
• Phagocytosis is triggered process: it requires activation of
receptors that transmit signals. The phagosome fuse with
lysosomes inside the cell and ingested material is degraded.
trigger is an antibody that binds to the infectious microorganisms
forming a coat that recognized by macrophages and neutrophils.
• Pinocytosis is constitutive: occurs continuously, regardless of the
cell needs.
Phagocytosis: a neutrophil reshaping a plasma membrane, forming a
pseudopod
Figure 13-47a Molecular Biology of the Cell (© Garland Science 2008)
Pseudopod formation requires reshaping of actin and PI3 kinase required
for closure of the phagosome.
Figure 13-47b Molecular Biology of the Cell (© Garland Science 2008)
Pinocytosis is continually occuring (cell size remains the same. So
endocytosios = exocytosis)
Formation of clathrin coated pit for processes of endocytosis
Figure 13-48 Molecular Biology of the Cell (© Garland Science 2008)
Not all pinocytosis vesicles are clathrin coated
Caveolae is needed
Figure 13-49 Molecular Biology of the Cell (© Garland Science 2008)
CAVEOLAE
• Form membrane microdomains – lipid rafts, rich in cholesterol and
glycosphingolipids and GPI-anchored membrane proteins
• Major protein in caveolae is caveolin – integral membrane protein
• Caveolae vesicles use Dynamin to pinch off
• Caveolins do not dissociate from formed vesicles, so they are
delivered to the target compartments.
RECEPTOR MEDIATED ENDOCYTOSIS
• Takes up specific macromolecules from the extracellular fluid.
• Macromolecules bind to specific receptors and are taken up by
clathrin coated vesicles.
• For example, cholesterol is taken up by receptor mediated
endocytosis ; if this is blocked then cholesterol will accumulate in
blood vessels causing atherosclerosis, plaques deposits that cause
strokes and heart attacks.
• Most Cholesterol is carried in blood as lipid-protein particle: low
density lipoprotein (LDL), when a cell needs cholesterol for
membrane synthesis it makes the receptor for LDL.
LDL particle
Figure 13-50 Molecular Biology of the Cell (© Garland Science 2008)
RECEPTOR MEDIATED ENDOCYTOSIS OF CHOLESTEROL:
vesicles are targeted to early endosomes, LDL is released  lysosomes,
here it gets hydrolyzed to make free cholesterol  cell uses it to make
membranes
Figure 13-51 Molecular Biology of the Cell (© Garland Science 2008)
Possible fates for transmembrane receptor proteins that have been
endocytosed
Figure 13-52 Molecular Biology of the Cell (© Garland Science 2008)
Receptor mediated endocytosis of LDL: EE as a sorting station
Figure 13-53 Molecular Biology of the Cell (© Garland Science 2008)
RECEPTOR MEDIATED ENDOCYTOSIS OF TRANSFERRIN
• Transferrin is a protein that binds to iron
• It is taken up by receptor-mediated endocytosis
• Both receptor and transferrin bound to iron go to EE
• Iron is released, and the receptor with transferrin are recycled back
to the PM.
• At the neutral pH of the extracellular fluid, the empty transferrin is
released.
• Clathrin-dependent receptor mediated endocytosis is highly
regulated: ubiquitin tagging, by mono-ub or multi-ub is needed.
(remember poly-ubiquitination – a long chain- targets for degradation
in proteosaome, it is different from mono (1 ub) or poly (many mono-)
that targets for receptor mediated endocytosis.
Figure 13-54 Molecular Biology of the Cell (© Garland Science 2008)
Details of endocytic pathway from PM to lysosomes
Figure 13-56 Molecular Biology of the Cell (© Garland Science 2008)
Figure 13-57 Molecular Biology of the Cell (© Garland Science 2008)
Skip any sections not included in this slide presentation
Receptors can be “stored” in recycling endosomes to be used when
needed (this is faster than having to express from the gene level)
Figure 13-61 Molecular Biology of the Cell (© Garland Science 2008)
Page 799 Molecular Biology of the Cell (© Garland Science 2008)
TWO PATHWAYS FOR EXOCYTOSIS : CONSTITUTIVE AND
REGULATED
Figure 13-63 Molecular Biology of the Cell (© Garland Science 2008)
The three best understood pathways for protein sorting in the trans
Golgi Network
Figure 13-64 Molecular Biology of the Cell (© Garland Science 2008)
Exocytosis of secretory vesicles
Figure 13-66a Molecular Biology of the Cell (© Garland Science 2008)
Figure 13-66b Molecular Biology of the Cell (© Garland Science 2008)
Secretory vesicles wait near the PM until signaled to release their
contents (such as Calcium entry into cells)
Figure 13-68 Molecular Biology of the Cell (© Garland Science 2008)
Examples of exocytosis leading to membrane enlargement
Figure 13-70 Molecular Biology of the Cell (© Garland Science 2008)
Exocytosis has to be targeted to the correct membrane domain
Figure 13-71 Molecular Biology of the Cell (© Garland Science 2008)
Two ways of sorting PM proteins in a polarized epithelial cell
Transcytosis
Figure 13-72 Molecular Biology of the Cell (© Garland Science 2008)
The formation of synaptic vesicles
Figure 13-73 Molecular Biology of the Cell (© Garland Science 2008)