Cell membrane
Lecture-8: Vesicular traffic (II)
Reference: Chapter 14
Lodish Harvey et al. (2008) Molecular Cell Biology (6th edition)
Publisher: W.H. Freeman and Company
Exocytosis
Constitutive and regulated secretion
25.7 Protein localization depends on further signals
Lysosomes are small bodies, enclosed
by membranes, that contain hydrolytic
enzymes in eukaryotic cells.
25.7 Protein
localization depends
on further signals
Figure 25.22 A transport
signal in a transmembrane cargo protein
interacts with an adaptor
protein.
25.7 Protein localization depends on further signals
Figure 25.23 A
transport signal
in a luminal
cargo protein
interacts with a
transmembrane
receptor that
interacts with an
adaptor protein.
Insulin is a good
example of a protein
that is stored in
secretory vesicles
until a cell receives
an signal to secrete
the insulin.
Processing to the final form
occurs in the secretory
vesicle.
Removal of the Presequence (not
shown), folding and
disulfide bond
formation occur in
ER.
This is an example of a protein
that you would not want to treat
with mercaptoethanol because
reduction of disulfide bonds
would inactivate the protein.
“pre-pro-proteins”
Some proteins are processed in secretory vesicles into multiple small polypeptides.
One explanation for this approach is that the small polypeptides are too short to be
cotranslationally transported into the ER.
25.7 Protein localization
depends on further signals
Figure 25.5 Processing for a complex
oligosaccharide occurs in the Golgi and
trims the original preformed unit to the
inner core consisting of 2 N-acetylglucosamine and 3 mannose residues.
Then further sugars can be added, in the
order in which the transfer enzymes are
encountered, to generate a terminal
region containing N-acetyl-glucosamine,
galactose, and sialic acid.
Modification of the N-linked oligosaccharides is done by enzymes
in the lumen of various Golgi compartments.
1. Sorting in TGN
2. Protection from protease digestion
3. Cell to cell adhesion via selectins
25.8 ER proteins
are retrieved from
the Golgi
Figure 25.24 An (artificial) protein
containing both lysosome and ERtargeting signals reveals a pathway
for ER-localization. The protein
becomes exposed to the first but not
to the second of the enzymes that
generates mannose-6-phosphate in
the Golgi, after which the KDEL
sequence causes it to be returned to
the ER.
25.8 ER proteins
are retrieved from
the Golgi
Figure 25.24 An (artificial)
protein containing both lysosome
and ER-targeting signals reveals
a pathway for ER-localization.
The protein becomes exposed to
the first but not to the second of
the enzymes that generates
mannose-6-phosphate in the
Golgi, after which the KDEL
sequence causes it to be returned
to the ER.
Endocytosed molecules that are
destined for the lysosome go from
the early endosome to the
multivesicular body to the late
endosome. Fusion of transport
vesicles carrying acid hydrolases
from the Golgi causes the late
endosome to mature into a
lysosome.
In some cases, both the
receptor and the ligand are
transported to the lysosome.
This is the case for EGF and
its receptor. EGF triggers a
cell to proliferate but the
signal is only required for a
short time. To limit the
response time both the
receptor and the ligand are
removed from the membrane.
Mosaic organization of endosomes: subdomains
Tubular-vesicular endosomes sort
membrane components from
lumenal components
Y YY Y Y
Y Y Y
Y
Y
Y
Y
Y
Y
Y Y
Experimental demonstration that internalized receptorligand complexes dissociate in endosomes
Hepatocyte:
Sorting of membrane from
contents: surface area to
volume ratio.
Narrow diameter
tubules
Asialglycoproteins a
their receptor.
Late Endosomes Contain Internal Vesicles
Maturation from early to late
endosomes occurs through the
formation of multivesicular bodies
(MVBs). The MVBs move deeper
into the cytoplasm fusing with each
other and pre-exisiting late
endosomes. These structures are
characterized by the formation of
internal vesicles.
Vesicles inside of vesicles.
Late Endosomes Sort By Selective Internalization
of Limiting Membrane
The formation of internal
vesicles by pinching off of
the limiting membrane of
MVBs/late endosomes is a
sorting process.
Membrane proteins destined
for degradation are marked
with a covalent monoubiquitin tag.
These mono-ubiquitinated
membrane proteins are
The Machinery for MVB formation is used by retroviruses
to bud
1. Ubiqutinated Hrs
protein on
the endosome recruits
Ub-tagged
TM cargo to buds then
recruits
ESCRT complexes.
2. ESCRT Required to
pinch off
internal vesicles.
3. The Vps4 ATPase
disassembles
ESCRT.
HIV Budding from the cell surface
Vesicle budding and fusion
 Coated vesicles are formed by polymerization of coat proteins onto a
membrane to form vesicle buds and then pinch off from the membrane
to release a complete vesicle.
 Vesicle budding is initiated by recruitment of a GTP-binding proteins:
- ARF protein is for both COPI and clathrin vesicles.
- Sar1 protein is for COPII vesicles.
 Vesicles fuse with its target membrane in a process involves interaction
of cognate SNARE proteins.
Vesicle budding
 Step 1: Soluble Sar1-GDP is converted to
Sar1-GTP by Sec12, a GEF on ER membrane.
Binding of GTP causes a conformational
change in Sar1 that exposes its hydrophobic
N-terminus, leading to the anchorage of Sar1
to the ER membrane.
 Step 2: Attached Sar1-GTP serves as a
binding site for the Sec23/Sec24 coat protein
complex (COPII subunits). Membrane cargo
proteins are recruited to the vesicle bud by
binding of sorting signal sequence.
 Step 3: Once vesicles are released, the
Sec23 subunit promotes Sar1 GTPase
activity and leads to GTP hydrolysis by Sar1.
 Step 4: Release of Sar1-GDP from the
vesicle membrane causes disassembly of
the COPII coat.
Sorting signals in cargo proteins
 For membrane cargo proteins, the vesicle coat selects these proteins
by directly binding to their cytoplasmic sorting signals on cytosolic
portion, while for soluble luminal proteins, the vesicle coat selects these
proteins by indirectly binding to their luminal sorting signals through a
cargo receptor.
•
Regulation of endocytosis. Several different
kinds of proteins and lipids regulate
internalization and endosomal sorting. Rab
proteins are membrane associated, Ras-like
GTPases that control membrane fusion. Different
Rabs are associated with particular endosomes.
Inositol phospholipids (phosphoinositides)
constitute a small fraction of the phospholipids in
the plasma membrane and endosomal membranes.
Distinct regions of the plasma membrane and
different endosomes are enriched in particular
varieties of phosphoinositides which bind with
different affinities to proteins with lipid-binding
domains. For example, the ENTH domain of
Epsin (see below) binds PI(4,5)P2, which is
enriched at the plasma membrane in vertebrate
cells. Some transmembrane proteins have
cytoplasmically located internalization signals
that are part of their primary amino acid sequence,
and these may bind AP-2. Alternatively, a
ubiquitin (Ub) polypeptide that serves as an
endocytosis signal may be added
posttranslationally to the cytoplasmic domain, and
these signals
The SNARE complex
 During exocytosis of secreted proteins, the v-SNARE is VAMP (vesicleassociated membrane protein). The t-SNAREs are syntaxin, an integral
membrane protein, and SNAP-25 which is attached to membrane by a
hydrophobic lipid anchor.
 The four helices (one from VAMP, one from syntaxin, and two from
SNAP-25) to coil around one another to form a four-helix bundle. The
stability of bundle is hold by the electrostatic interactions of oppositecharged amino acids between helices.
 The dissociation of SNARE complexes requires energy and two proteins,
NSF (NEM-sensitive factor) and α-SNAP (soluble NSF attachment
protein). NSF associates with a SNARE complex with the aid of α-SNAP,
which hydrolyzes ATP and releases energy to dissociate SNARE complex.
v-SNARE
(VAMP)
t-SNARE
(Syntaxin)
t-SNARE
(SNAP-25)
Vesicles ducking and fusion
 Step 1: The ducking between the vesicle and the
target membrane is mediated by the interaction
between the vesicle-attached Rab GTPase and its
effector on the target membrane.
 Step 2: VAMP proteins on the vesicle surface
interact with the cytosolic domains of syntaxin
and SNAP-25 on the target membrane to form
a coiled-coil SNARE complex, which brings
two membranes close together.
 Step 3: Membrane fusion immediately after the
formation of SNARE complex.
 Step 4: NSF associating with α-SNAP binds
to the SNARE complexes. The NSF-catalyzed
hydrolysis of ATP then drives disassembly of
the SNARE complexes. At the same time,
Rab-GTP is hydrolyzed to Rab-GDP and
dissociates from the Rab effector.
Vesicle trafficking between ER and cis-Golgi
 Step 1-3: the anterograde transport
from the ER to cis-Golgi is mediated
by COPII vesicles. These vesicles
contain newly synthesized proteins
destined for the Golgi, cell surface or
lysosome.
 Step 4-6: the retrograde transport
from the cis-Golgi to ER is mediated
by COPI vesicles. The purpose of
this transport is to retrieve v-SNAREs,
membranes and misfolded proteins
back to the ER.
KDEL receptor in retrograde transport
 Most soluble ER-resident proteins carry
a Lys-Asp-Glu-Leu (KDEL) sequence at
their C-terminus, forming KDEL sorting
signal.
 The KDEL sorting signal is recognized
and bound by the KDEL receptor which
is located mainly in the cis-Golgi and in
both COPII and COPI vesicles.
 The binding affinity of KDEL receptor is
enhanced at low pH. Thus, the difference
in the pH of the ER and Golgi favors
binding of KDEL-bearing proteins to the
receptor in Golgi-derived vesicles and
their release in the ER.
 This retrieval system prevents depletion
of ER luminal proteins such as chaperone
proteins.
Models for the polarization of the Golgi
 In the vesicular transport model, the Golgi cisternae are static
organelles, which contain their resident proteins. The passing of
molecules from cis to trans through anterograde transport.
 In the cisternal maturation model, the Golgi cisternae are dynamic
organelles. Each cisterna matures as it migrates forward. At each
stage, the Golgi-resident proteins carried forward in a cisterna are
moved backward to an earlier compartment by retrograde transport.
Tight junctions divide the PM of
polarized cells into domains
•
•
•
Apicobasal Polarity is associated
with many cell-types.
Epithelial cells form ion-tight
monolayers of high electrical
resistance.
Apical and Basolateral Domains
are different in Lipid and Protein
Composition
Polarized Cells
Membrane trafficking is critical to Polarity
• Sorting at the TransGolgi
• Retention After
Secretion
• Sorting After
Endocytosis
• Sorting Signals
Basolateral:
Tyrosine or
DiLeucine
Apical:
N or O-linked
Glycosylation
Or TM domain
Three Destinations After
Endocytosis In a Polarized Cell
Polarized Epithelia Have Apical and
Basolateral Specific Endosomes
• The additional
complexity of
the plasma
membrane
requires extra
endosomal
compartments
for sorting.
Basolateral Targeting and
Human Disease
Koivisto et al., 2001: In the familial
hypercholesterolemia (FH)-Turku LDL
receptor allele, a mutation of glycine 823
residue affects the signal required for
basolateral targeting in MDCK cells. We
show that the mutant receptor is
mistargeted to the apical surface in both
MDCK and hepatic epithelial cells,
resulting in reduced endocytosis of LDL
from the basolateral/sinusoidal surface.
This work suggests that a defect in
polarized LDL receptor expression in
hepatocytes underlies the
hypercholesterolemia in patients
harboring this allele.
QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.
Processing of N-linked glycosylation in Golgi
 The Golgi complex is organized into
3-4 cisternae, which contain different
enzymes for protein glycosylation.
 N-linked glycosylation in the Golgi:
> In cis-Golgi, three mannose residues
are removed (1).
> In medial-Golgi, three GlcNAc (2,4)
and one fucose (5) residues are added,
while two mannose (3) residues are
removed.
> In trans-Golgi, three galactose (6)
residues are added, followed by the
linkage of N-Acetylneuraminic acid (7)
on each galactose residue.
(GlcNAc)
 Each enzyme move dynamically from
the later to the earlier cisterna through
retrograde vesicle transports.
Evidence of Golgi cisternal maturation
 Yeast cells expressing:
> the cis-Golgi protein Vrg4-GFP (green)
> the trans-Golgi protein Sec7-DsRed (red)
 A compartment rarely contains both cis- and trans-Golgi
proteins at the same time.
Endocytosis
• Why do cells need endocytosis?
• Is there more than one endocytic pathway ?
–
–
–
–
Clathrin-mediated uptake
Caveolae
Non-clathrin/non-caveolae pathways
Pinocytosis/ Phagocytosis
• What are the functional consequences of endocytosis?
• How are endocytic structures formed and how do they
know where to go?
• Where do the textbook models come from?
Is cholera toxin internalized to the Golgi complex by a
clathrin-dependent process?
• Epsin and eps15 mutants
inhibit clathrin-mediated
transferrin (Tf) uptake to
recycling endosomes
• Epsin and eps15 mutants do
not affect cholera toxin Bsubunit (CTXB) uptake to
the Golgi complex (marked
by b-COP)
• Suggests CTXB is delivered
to the Golgi complex by a
clathrin-independent
pathway
b-COP: Marker for
the Golgi complex
Does internalized CTXB
pass through early
endosomes?
Nichols et al. 2001 J. Cell Biol.
• Early endosome function
requires the GTPase Rab5
• Dominant negative rab5
S34N (GDP bound)
expression perturbs early
endosomes and blocks
transferrin uptake
• Rab5 S34N does not affect
delivery of CTXB to the
Golgi complex
• Suggests CTXB does not
pass through early
endosomes
Active Membrane Transport – Review
Process
Energy Source
Example
Active transport of solutes
ATP
Movement of ions across
membranes
Exocytosis
ATP
Neurotransmitter secretion
Endocytosis
ATP
White blood cell phagocytosis
Fluid-phase endocytosis
ATP
Absorption by intestinal cells
Receptor-mediated
endocytosis
ATP
Hormone and cholesterol
uptake
Endocytosis via caveoli
ATP
Cholesterol regulation
Endocytosis via coatomer
vesicles
ATP
Intracellular trafficking of
molecules
The endocytic pathway is divided into the
early endosomes and late endosomes pathway.
Materials in the early endosomes are sorted:
 Integral membrane proteins are shipped back to the
membrane;
 Other dissolved materials and bound ligands
Multivesicular body (MT mediated transport) the
late endosomes.
Dissociation of internalized ligand-receptor complexs
in the late endosomes. Molecules that reach the late
endosomes are moved to lysosomes.
The macromolecules that are degraded in the lysosome arrive by
endocytosis, phagocytosis, or autophagy.
lysosomes
 Lysosomes contain about 40 types of hydrolytic enzymes. For optimal
activity, they need to be activated by proteolytic cleavage and an acidic
environment, which is established by the V-class H+ pumps on lysosomal
membrane.
 Mature endosomes containing numerous vesicles in their interior are
usually called multivesicular endosomes. Fusion of a multivesicular
endosome directly with a lysosome releases the internal vesicles into
the lumen of the lysosome, where they can be degraded.
 Lysosomal membrane proteins are not incorporated into internal
endosomal vesicles, thus keeping them away from degradation.
Formation of multivesicular endosomes
 Proteins destined to the multivesicular endosome are tagged with
ubiquitin at the plasma membrane, the TGN or the endosomal membrane.
 In the endosomal budding, a ubiquitin-tagged Hrs protein on the
endosomal membrane facilitates loading of ubiquitinated cargo proteins
into vesicle buds and then recruits cytosolic ESCRT proteins to the
membrane (step 1).
 The membrane-associated ESCRT proteins act to complete vesicle
budding, leading to
release of a vesicle
carrying cargo into the
endosome (step 2).
 ESCRT proteins are
disassembled by the
ATPase Vps4 and
returned to the cytosol
(step 3).
Apical-basolateral protein sorting
 Proteins destined for either the apical or the basolateral membranes are
sorted in the TGN into different transport vesicles.
 When cells are infected with VSV and influenza viruses simultaneously,
the VSV G glycoprotein is found only on the basolateral membrane,
whereas the influenza HA glycoprotein is found only on the apical
membrane.
 In hepatocytes, membrane proteins
are directed first to the basolateral
membrane. Then, both apical and
basolateral proteins are
endocytosed in the same vesicles:
> the basolateral proteins are
recycled back to basolateral
membrane.
> the apical proteins are
transported across the
cell to apical membrane
(transcytosis).
Intracellular Vesicular Transport
Vesicular transport of neurotransmitters
Neurotransmission (Latin: transmissio = passage, crossing; from transmitto = send, let through), also called synaptic transmission, is an
electrical movement within synapses caused by a propagation of nerve impulses. As each nerve cell receives neurotransmitter from the
presynaptic neuron, or terminal button, to the postsynaptic neuron, or dendrite, of the second neuron, it sends it back out to several neurons, and
they do the same, thus creating a wave of energy until the pulse has made its way across an organ or specific area of neurons.
Nerve impulses are essential for the propagation of signals. These signals are sent to and from the central nervous system via efferent and afferent
neurons in order to coordinate smooth, skeletal and cardiac muscles, bodily secretions and organ functions critical for the long-term survival of
multicellular vertebrate organisms such as mammals.
Neurons form networks through which nerve impulses travel. Each neuron receives as many as 15,000 connections from other neurons. Neurons
do not touch each other; they have contact points called synapses. A neuron transports its information by way of a nerve impulse. When a nerve
impulse arrives at the synapse, it releases neurotransmitters, which influence another cell, either in an inhibitory way or in an excitatory way. The
next neuron may be connected to many more neurons, and if the total of excitatory influences is more than the inhibitory influences, it will also
"fire", that is, it will create a new action potential at its axon hillock, in this way passing on the information to yet another next neuron, or
resulting in an experience or an action.
An example of propagation among neurons is the heart beat. A beat is made when a signal is sent from the Sinoatrial node in a sequence that
causes the heart to fully contract emptying all the blood in it and refilling with all new blood. It is important that the pulse is sent out from the SA
node because the direction of the pulse between the neurons is what drives the muscle to fully contract. If the pulse comes in from the AV node
the heart will stutter and not empty all the blood into the body.
Synaptic vesicle and plasma membrane proteins important for
vesicle docking and fusion
Lodish et al. Figure 21-31
6. Membrane Potentials and Nerve Impulses
A. K+ gradients maintained by the Na+-K+ ATPase are responsible for
the resting membrane potential.
B. The action potential: The changes in
ion channels and membrane potential.
Resting state: All Na+ and K+ channels
closed.
Depolarizing phase: Na+ channels
open,triggering an action potential.
Repolarizing phase: Na+ channels
inactivated, K+ channels open.
Hyperpolarizing phase: K+ channels
remain open, Na+ channels inactivated.
The sequence of events during synaptic transmission:
Excitable membranes exhibit “all-or-none” behavior.
Propagation of action potentials as an impulse.
Cycling of neurotransmitters and synaptic vesicles
Cycling of neurotransmitters and synaptic vesicles
 The uncoated vesicles employ a variety of antiporters (blue) to import
neurotransmitters (transmitters) from cytosol (step 1).
 Transmitter-loaded vesicles move to the active zone (step 2).
 Vesicle docks on the membrane of a presynaptic cells, which is mediated
by SNAREs. Synaptotagmin, a Ca+2 sensor for exocytosis of transmitter,
prevents membrane fusion (step 3).
 In response to an action potential, voltage-gated Ca+2 channels in
membrane open, allowing an influx of Ca+2 from the synaptic cleft. It
causes a conformational change in synaptotagmin, leading to fusion of
docked vesicles with plasma membrane and release of transmitters into
the synaptic cleft (step 4).
 After clathrin/AP vesicles containing v-SNARE and transmitter transporter
proteins bud inward and are pinched off in a dynamin-mediated process,
they loss their coat proteins. At the same time, Na+-transmitter symporters
take up transmitter from the synaptic cleft (step 5).
 Vesicles are recovered by endocytosis, creating uncoated vesicles (step
6).
25.6 Budding and
fusion reactions
Figure 25.16 A
SNAREpin forms
by a 4-helix bundle.
Photograph kindly
provided by Axel
Brunger.
25.6 Budding
and fusion
reactions
Figure 25.17 A SNAREpin complex protrudes parallel to the plane of the
membrane. An electron micrograph of the complex is superimposed on the
model. Photograph kindly provided by James Rothman.
25.6 Budding and
fusion reactions
Figure 25.18 Neurotransmitters
are released from a donor
(presynaptic) cell when an
impulse causes exocytosis.
Synaptic (coated) vesicles fuse
with the plasma membrane, and
release their contents into the
extracellular fluid.
25.6
Budding
and
fusion
reactions
Figure 25.19 The kiss and run model proposes that a synaptic
vesicle touches the plasma membrane transiently, releases its
contents through a pore, and then reforms.
25.6 Budding and
fusion reactions
Figure 25.20 When synaptic
vesicles fuse with the plasma
membrane, their components
are retrieved by endocytosis
of clathrin-coated vesicles.
25.6 Budding and
fusion reactions
Figure 25.21 Rab
proteins affect
particular stages of
vesicular transport.
Endocytosis is a process by which cells take up substances by invaginating the
plasma membrane. This process can capture both membrane bound and soluble
components.
There are several subclasses of endocytosis:
•Phagocytosis takes up large particles and cells.
•Pinocytosis continuously takes up small amounts of fluid.
•Receptor-mediated endocytosis selectively takes up membrane receptors
and associated ligands.
Endocytosis takes up large amounts of the plasma membrane and is balanced by
the return of membrane components to the plasma membrane by exocytosis.
• GenMAPP-generated Ras/ERK signaling
pathway shaded to correspond with gene
expression data. CAV1=caveolin 1;
CAV2=caveolin 2; ER=estrogen receptor
Problem based learning
Exocytosis: Material (wastes etc.) are expelled from the cell (recall golgi vesicles).
Secretory vesicles concentrate
and store products. Secreted
products can be either small
molecules or proteins. Proteins
originate at the ER. In the
Golgi, these proteins aggregate
and are packaged into transport
vesicles as aggregates.
Exocytosis and endocytosis
 Exocytosis: a process that a cell releases intracellular molecules
(such as hormones, secretory proteins) contained within a
membrane-bounded vesicle by fusion of the vesicle with its plasma
membrane.
 Endocytosis: a process that a cell uptake extracellular material by
engulfing it within cell, including receptor-mediated endocytosis,
phagocytosis and pinocytosis.
 Vesicular transport: transport vesicles carrying material as cargo bud
off from the donor compartment and fuse with the target compartment.
Vesicular Transport: Exocytosis
• Secreting material or replacement of plasma
membrane
Introduction
Figure 25.2 Vesicles are
released when they bud
from a donor
compartment and are
surrounded by coat
proteins (left).
During fusion, the coated
vesicle binds to a target
compartment, is uncoated,
and fuses with the target
membrane, releasing its
contents (right).
Exocytosis
• Vesicle moves to cell surface
• Membrane of vesicle fuses
• Materials expelled orCell
discharges material
• Reverse of endocytosis
•
Exocytosis (post-Golgi trafficking)
• Where do newly synthesized membrane and secretory
proteins need to go and how do they get there?
– Secretion (constitutive and regulated)
– PM protein delivery (polarized and non-polarized cells)
– Lysosomal targeting
• How are proteins packaged into vesicles, and how do
the vesicles know where to go?
• What do we know about how the Golgi complex
actually works?
• Where do the textbook models come from?
Exocytosis (post-Golgi trafficking)
• Where do newly synthesized membrane and
secretory proteins need to go and how do they get
there?
– Secretion (constitutive and regulated)
– PM protein delivery (polarized and non-polarized cells)
– Lysosomal targeting
• How are proteins packaged into vesicles, and how do
the vesicles know where to go?
• What do we know about how the Golgi complex
actually works?
• Where do the textbook models come from?
Overview of the secretory/exocytic pathway
Recycling vesicles
Transitional
ER site
COPI vesicles
To plasma membrane
To secretory granules
COPII
vesicles
TGN = trans-Golgi network
cis
medial
trans
TGN
To endosomes
Regulated secretion
• Occurs in endocrine,
exocrine and neuronal
cells
– Insulin secretion in
pancreatic b-cells
– Trypsinogen secretion
in pancreatic acinar
cells
• Exocytosis occurs in
response to a trigger
(ex. Ca2+)
5-48
Processing of regulated secretory proteins
•
•
•
•
Proteins undergo proteolytic
processing from a proprotein to the
mature form
Processing occurs in secretory
vesicles as they move away from the
TGN
Undergo selective aggregation with
one another that aids in their sorting
Proteins become highly concentrated
(condensed)  dense-core granules
Insulin in the regulated secretory pathway
Antibody binds proinsulin (not
insulin)
Mature
secretory
vesicles
Antibody binds insulin (not
proinsulin)
Mature
secretory
vesicles
Clathrin coat
Golgi complex
Golgi complex
Immature
secretory
vesicles
Vesicle
budding
from TGN
Immature
secretory
vesicles
Vesicle
budding
from TGN
17-41
Constitutive secretion/ exocytosis of plasma
membrane proteins
• Delivered via membrane
vesicles directly from the TGN
to the cell surface
• Share same vesicles as
constitutively secreted proteins
• Remarkably little is known
about how plasma membrane
proteins are sorted into
secretory vesicles
• May be more than one class of
carrier vesicles
VSVGts045, a model protein for
studying the secretory pathway (shown
here tagged with GFP)
Visualizing the secretory pathway:
Fusion of TGN-derived vesicles containing VSVGts045-CFP or YFP-GLGPI with the plasma membrane observed in living cells using total
internal reflection microscopy
Keller et al (2001) Nature Cell Biol.
Exocytosis in polarized epithelial cells
• The functions and thus protein
composition of the apical and
basolateral domain differ
• Proteins and lipids must be
delivered to the correct PM
domain
• Proteins can be sorted directly
from the TGN to the apical or
basolateral domain
• Proteins can also be delivered
indirectly by transcytosis
Mostov et al. 1999 Cell 99:121-122
“ Direct” versus “indirect” (transcytotic) trafficking
in polarized cells
Lodish et al. Figure 17-43
Transcytosis
In the infant intestine, antibodies are
ingested from mother’s milk.
They bind to Fc receptors on the
apical surface of the intestine.
The IgG-FcR complex is
transcytosed to the basolateral side
where the IgG is released.
The empty FcR is then transcytosed
back to the apical side.
The pH values on either side of the
epithelium are critical for correct
binding and release.
Transcytosis provides a way to deliver proteins across an
epithelium.
Transport of antibodies in
milk across the gut
epithelium of baby rats.
Acidic pH of the gut favor
association of antibody with
Fc receptor whereas the
neutral pH of the
extracellular fluid favors
dissociation.
Transcytosis: a closer look
lumen
– Contains sorting information in its
cytoplasmic tail
– pIgA is secreted into the the gut
lumen, bile and milk as part of the
mucosal immune response
pIgA-R
pIgA
• Transcytosis: transport of
macromolecular cargo from
one side of the cell to the other
• Transcytosis is also utilized in
the biosynthetic trafficking of
some PM proteins
• pIgA-receptor is a model for
studying transcytosis
Blood/interstitial
synthesizes IgA
Tuma and Hubbard (2003)
Ras trafficking via the
secretory pathway
HRas
KRas
• Ras is targeted to the plasma
membrane by its C-terminal
domain
– CAAX
– Second signal (polybasic or
palmitoylation)
• The CAAX motif targets the
protein to the ER
• PM delivery of HRas but not
KRas is blocked by BFA
• Indicates HRas relies on
vesicular transport to reach
the cell surface
Magee and Marshall (1999) Cell
Blocks to secretion/ PM protein delivery
• 20º C (mechanism unknown, but it works!)
• Brefeldin A (inhibits assembly of COPI vesicles;
blocks ER-to-Golgi trafficking)
• Cholesterol depletion (disrupts lipid rafts)
• Sec mutants (yeast)
• Microinjection of antibodies against regulatory
proteins
25.7 Protein localization depends on further signals
Lysosomes are small bodies, enclosed
by membranes, that contain hydrolytic
enzymes in eukaryotic cells.
Lysosomal trafficking
• Trafficking of soluble lysosomal hydrolases
– Hydrolases are modified by mannose-6-phosphate (M6P) in the cis-Golgi
– The M6P receptor captures the hydrolases in the TGN as the receptor
cycles between the TGN and late endosomes in clathrin-coated vesicles
(AP-1, GGA)
– The phosphate is removed from hydrolases in late endosomes to prevent
recycling of the hydrolases with the M6P receptor
– Secreted hydrolases are captured and delivered to lysosomes by
endocytosis via PM-localized M6P receptors
• Trafficking of lysosomal membrane proteins
– Sorting information is contained in their cytoplasmic tails
The Lysosome
The endpoint of the
endocytosis pathway for
many molecules is the
lysosome, a highly acidic
organelle rich in
degradative enzymes.
The V-ATPase maintains
the high acidity of the
lumen by pumping protons
across the lipid bilayer.
Trafficking of lysosomal hydrolases to lysosomes by the
mannose-6-phosphate receptor
Exocytosis (post-Golgi trafficking)
• Where do newly synthesized membrane and secretory
proteins need to go and how do they get there?
– Secretion (constitutive and regulated)
– PM protein delivery (polarized and non-polarized cells)
– Lysosomal targeting
• How are proteins packaged into vesicles, and how
do the vesicles know where to go?
• What do we know about how the Golgi complex
actually works?
• Where do the textbook models come from?
Key steps in the formation of clathrin-coated vesicles
Activation
(TGN)
Coat assembly
Activation (PM)
Scission
Cargo capture
Uncoating
Kirchhausen 2000 Nature Reviews Molecular Cell Biology 1:187
Making and moving vesicles: general sorting and
trafficking machinery
• Cargo sorting signals
• Membrane lipids
• Vesicle formation- clathrin and
accessory proteins
• Cargo capture- adaptors
• “Pinchase”- dynamin
• Direct vesicle movement- actin,
microtubules and motors
• Vesicle targeting and fusion
machinery- Rabs, SNARES
• Docking sites on the plasma
membrane in polarized cellsexocyst
Lodish et al. Figure 17-51
Sorting signals in cargo molecules
Signal
sequence
Type of
protein
Transport
step
Vesicle type
Signal
receptor
Mannose-6phosphate
Secreted
(lysosomal)
TGN to PM
and late
endosome
clathrin
M6P-R, AP1
and AP2
Tyr-X-X-Ø
membrane
(endosome,
BL)
PM to
endosome
clathrin
AP2, AP1B
Leu-Leu (LL)
membrane
(endosome,
BL)
PM to
endosome
clathrin
AP2, AP1B
Selective
aggregation
secreted
(regulated)
TGN to
secretory
granule
clathrin
?
GPI-anchor
membrane
(apical)
TGN to PM
unknown
Lipid rafts/?
Exocytosis (post-Golgi trafficking)
• Where do newly synthesized membrane and secretory
proteins need to go and how do they get there?
– Secretion (constitutive and regulated)
– PM protein delivery (polarized and non-polarized cells)
– Lysosomal targeting
• How are proteins packaged into vesicles, and how do
the vesicles know where to go?
• What do we know about how the Golgi complex
actually works?
• Where do the textbook models come from?
The Golgi complex
3D EM tomography of the
Golgi complex
• Central organelle of the secretory
pathway
• Comprises stacks of flattened
cisternae
• Contains resident enzymes that
modify newly synthesized proteins
and lipids (ex. glycosylation)
• At the trans most stack, proteins
are sorted for delivery inside the
cell or for secretion
• Golgi morphology and
composition is maintained despite
the flux of proteins and lipids in
the secretory pathway
Nothing is simple when it comes to the Golgi complex
• How does cargo move through
the Golgi complex?
– Cisternal maturation vs vesicular
transport
• How is the Golgi complex
inherited during mitosis?
– ER absorption vs vesiculation
• How does the Golgi complex
form?
– Self-organizes following ER
export vs. stable matrix which
nucleates formation
Models for transport through the Golgi
Vesicular transport
Cisternal
maturation
Interlinked
network
Elsner et al 2003
What is the fate of the Golgi in mitosis?
Barr, 2004
Exocytosis (post-Golgi trafficking)
• Where do newly synthesized membrane and
secretory proteins need to go and how do they get
there?
– Secretion (constitutive and regulated)
– PM protein delivery (polarized and non-polarized cells)
– Lysosomal targeting
• How are proteins packaged into vesicles, and how do
the vesicles know where to go?
• What do we know about how the Golgi complex
actually works?
• Where do the textbook models come from?
25.10 Summary
1. Proteins that reside within the reticuloendothelial system or that are
secreted from the plasma membrane enter the ER by cotranslational
transfer directly from the ribosome.
2. Proteins are transported between membranous surfaces as cargoes in
membrane-bound coated vesicles.
3. Modification of proteins by addition of a preformed oligosaccharide
starts in the endoplasmic reticulum.
4. Different types of vesicles are responsible for transport to and from
different membrane systems.
5. COP-I-coated vesicles are responsible for retrograde transport from the
Golgi to the ER.
6. COP-II vesicles undertake forward movement from the ER to Golgi.
25.10 Summary
7. In the pathway for regulated secretion of proteins, proteins are
sorted into clathrin-coated vesicles at the Golgi trans face.
8. Budding and fusion of all types of vesicles is controlled by a
small GTP-binding protein.
9. Vesicles recognize appropriate target membranes because a
vSNARE on the vesicle pairs specifically with a tSNARE on the
target membrane.
10. Receptors may be internalized either continuously or as the
result of binding to an extracellular ligand.
11. The acid environment of the endosome causes some receptors
to release their ligands; the ligand are carried to lysosomes, where
they are degraded, and the receptors are recycled back to the plasma
membrane by means of coated vesicles.
A simple experiment shows that many sorting signals consist of a
continuous stretch of amino acid sequence called a “signal
sequence”
Fusing sorting signals to GFP
is particularly good way to do
this experiment.
GFP
Cytoplasmic
Nuclear
PAX-GFP
Actin-GFP