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Lecture-8: Vesicular traffic (II)Lecture-8: Vesicular traffic (II)
Cell membraneCell membrane
Reference: Chapter 14Reference: Chapter 14 Lodish Harvey et al. (2008) Molecular Cell Biology (6Lodish Harvey et al. (2008) Molecular Cell Biology (6thth edition) edition) Publisher: W.H. Freeman and Company Publisher: W.H. Freeman and Company
Exocytosis
Constitutive and regulated secretion
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 trans- membrane 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.
25.7 Protein localization depends on further signals
Removal of the Pre-sequence (not shown), folding and disulfide bond formation occur in ER.
Processing to the final form occurs in the secretory vesicle.
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.
Insulin is a good example of a protein that is stored in secretory vesicles until a cell receives an signal to secrete the insulin.
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.
“pre-pro-proteins”
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-acetyl-glucosamine 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.
25.7 Protein localization depends on further signals
Modification of the N-linked oligosaccharides is done by enzymes in the lumen of various Golgi compartments.
1. Sorting in TGN2. Protection from protease digestion3. Cell to cell adhesion via selectins
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.
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.
25.8 ER proteins are retrieved from
the Golgi
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
YY Y
Y
Y
YY
YY Y Y
Y Y Y Y Y
Experimental demonstration that internalized receptor-ligand complexes dissociate in endosomes
Narrow diameter tubules
Hepatocyte:
Asialglycoproteins and their receptor.
Sorting of membrane fromcontents: surface area tovolume ratio.
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 mono-ubiquitin tag.
These mono-ubiquitinated membrane proteins are sorted into invaginating buds that pinch off into internal vesicles.
Internal vesicles and their contents are degraded in the lysosome.
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.
4. Ub-Gag mimics HrsRecruiting ESCRT to
HIVPM buds.
5. ESCRT pinches off buds
Releasing free virus, and Vps4
Recycles ESCRT.
HIV Budding from the cell surface
Vesicle budding and fusionVesicle budding and fusion
Vesicle budding is initiated by recruitment of a GTP-binding proteins: Vesicle budding is initiated by recruitment of a GTP-binding proteins: - ARF protein is for both COPI and clathrin vesicles.- ARF protein is for both COPI and clathrin vesicles. - Sar1 protein is for COPII vesicles.- Sar1 protein is for COPII vesicles.
Coated vesicles are formed by polymerization of coat proteins onto a Coated vesicles are formed by polymerization of coat proteins onto a membrane to form vesicle buds and then pinch off from the membrane membrane to form vesicle buds and then pinch off from the membrane to release a complete vesicle. to release a complete vesicle.
Vesicles fuse with its target membrane in a process involves interaction Vesicles fuse with its target membrane in a process involves interaction of cognate SNARE proteins.of cognate SNARE proteins.
Vesicle buddingVesicle budding
Step 4: Release of Sar1-GDP from the Step 4: Release of Sar1-GDP from the vesicle membrane causes disassembly of vesicle membrane causes disassembly of the COPII coat.the COPII coat.
Step 1: Soluble Sar1-GDP is converted to Step 1: Soluble Sar1-GDP is converted to Sar1-GTP by Sec12, a GEF on ER membrane. Sar1-GTP by Sec12, a GEF on ER membrane. Binding of GTP causes a conformational Binding of GTP causes a conformational change in Sar1 that exposes its hydrophobic change in Sar1 that exposes its hydrophobic N-terminus, leading to the anchorage of Sar1N-terminus, leading to the anchorage of Sar1 to the ER membrane.to the ER membrane.
Step 2: Attached Sar1-GTP serves as a Step 2: Attached Sar1-GTP serves as a binding site for the Sec23/Sec24 coat protein binding site for the Sec23/Sec24 coat protein complex (COPII subunits). Membrane cargo complex (COPII subunits). Membrane cargo proteins are recruited to the vesicle bud by proteins are recruited to the vesicle bud by binding of sorting signal sequence.binding of sorting signal sequence.
Step 3: Once vesicles are released, the Step 3: Once vesicles are released, the Sec23 subunit promotes Sar1 GTPase Sec23 subunit promotes Sar1 GTPase activity and leads to GTP hydrolysis by Sar1.activity and leads to GTP hydrolysis by Sar1.
Sorting signals in cargo proteinsSorting signals in cargo proteins For membrane cargo proteins, the vesicle coat selects these proteins For membrane cargo proteins, the vesicle coat selects these proteins by directly binding to their by directly binding to their cytoplasmic sorting signalscytoplasmic sorting signals on cytosolic on cytosolic portion, while for soluble luminal proteins, the vesicle coat selects these portion, while for soluble luminal proteins, the vesicle coat selects these proteins by indirectly binding to their proteins by indirectly binding to their luminal sorting signalsluminal sorting signals through a through a cargo receptor.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 complexThe SNARE complex
The four helices (one from VAMP, one from syntaxin, and two from 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 SNAP-25) to coil around one another to form a four-helix bundle. The stability of bundle is hold by the electrostatic interactions of opposite- stability of bundle is hold by the electrostatic interactions of opposite- charged amino acids between helices.charged amino acids between helices.
During exocytosis of secreted proteins, the v-SNARE is VAMP (During exocytosis of secreted proteins, the v-SNARE is VAMP (vvesicle-esicle- aassociated ssociated mmembrane embrane pprotein). The t-SNAREs are syntaxin, an integral rotein). The t-SNAREs are syntaxin, an integral membrane protein, and SNAP-25 which is attached to membrane by a membrane protein, and SNAP-25 which is attached to membrane by a hydrophobic lipid anchor.hydrophobic lipid anchor.
t-SNARE(SNAP-25)
t-SNARE(Syntaxin)
v-SNARE(VAMP)
The dissociation of SNARE complexes requires energy and two proteins, The dissociation of SNARE complexes requires energy and two proteins, NSF (NSF (NNEM-EM-ssensitive ensitive ffactor) and α-SNAP (actor) and α-SNAP (ssoluble oluble NNSF SF aattachment ttachment pprotein). NSF associates with a SNARE complex with the aid of α-SNAP, rotein). NSF associates with a SNARE complex with the aid of α-SNAP, which hydrolyzes ATP and releases energy to dissociate SNARE complex.which hydrolyzes ATP and releases energy to dissociate SNARE complex.
Step 4: NSF associating with α-SNAP binds Step 4: NSF associating with α-SNAP binds to the SNARE complexes. The NSF-catalyzed to the SNARE complexes. The NSF-catalyzed hydrolysis of ATP then drives disassembly of hydrolysis of ATP then drives disassembly of the SNARE complexes. At the same time, the SNARE complexes. At the same time, Rab-GTP is hydrolyzed to Rab-GDP and Rab-GTP is hydrolyzed to Rab-GDP and dissociates from the Rab effector.dissociates from the Rab effector.
Step 2: VAMP proteins on the vesicle surface Step 2: VAMP proteins on the vesicle surface interact with the cytosolic domains of syntaxin interact with the cytosolic domains of syntaxin and SNAP-25 on the target membrane to form and SNAP-25 on the target membrane to form a coiled-coil SNARE complex, which brings a coiled-coil SNARE complex, which brings two membranes close together.two membranes close together.
Step 3: Membrane fusion immediately after the Step 3: Membrane fusion immediately after the formation of SNARE complex.formation of SNARE complex.
Step 1: The ducking between the vesicle and the Step 1: The ducking between the vesicle and the target membrane is mediated by the interaction target membrane is mediated by the interaction between the vesicle-attached Rab GTPase and its between the vesicle-attached Rab GTPase and its effector on the target membrane.effector on the target membrane.
Vesicles ducking and fusionVesicles ducking and fusion
Vesicle trafficking between ER and Vesicle trafficking between ER and ciscis-Golgi-Golgi
Step 1-3: the anterograde transport Step 1-3: the anterograde transport from the ER to from the ER to ciscis-Golgi is mediated -Golgi is mediated by COPII vesicles. These vesicles by COPII vesicles. These vesicles contain newly synthesized proteins contain newly synthesized proteins destined for the Golgi, cell surface or destined for the Golgi, cell surface or lysosome.lysosome.
Step 4-6: the retrograde transport Step 4-6: the retrograde transport from the from the ciscis-Golgi to ER is mediated -Golgi to ER is mediated by COPI vesicles. The purpose of by COPI vesicles. The purpose of this transport is to retrieve v-SNAREs, this transport is to retrieve v-SNAREs, membranes and misfolded proteins membranes and misfolded proteins back to the ER.back to the ER.
KDEL receptor in retrograde transport KDEL receptor in retrograde transport
The KDEL sorting signal is recognized The KDEL sorting signal is recognized and bound by the KDEL receptor which and bound by the KDEL receptor which is located mainly in the is located mainly in the ciscis-Golgi and in -Golgi and in both COPII and COPI vesicles.both COPII and COPI vesicles.
This retrieval system prevents depletion This retrieval system prevents depletion of ER luminal proteins such as chaperone of ER luminal proteins such as chaperone proteins.proteins.
Most Most soluble ER-resident proteinssoluble ER-resident proteins carry carry a Lys-Asp-Glu-Leu (KDEL) sequence at a Lys-Asp-Glu-Leu (KDEL) sequence at their C-terminus, forming KDEL sorting their C-terminus, forming KDEL sorting signal.signal.
The binding affinity of KDEL receptor is The binding affinity of KDEL receptor is enhanced at low pH. Thus, the difference enhanced at low pH. Thus, the difference in the pH of the ER and Golgi favors in the pH of the ER and Golgi favors binding of KDEL-bearing proteins to the binding of KDEL-bearing proteins to the receptor in Golgi-derived vesicles and receptor in Golgi-derived vesicles and their release in the ER.their release in the ER.
Models for the polarization of the GolgiModels for the polarization of the Golgi
In the cisternal maturation model, the Golgi cisternae are dynamic In the cisternal maturation model, the Golgi cisternae are dynamic organelles. Each cisterna matures as it migrates forward. At each organelles. Each cisterna matures as it migrates forward. At each stage, the Golgi-resident proteins carried forward in a cisterna are stage, the Golgi-resident proteins carried forward in a cisterna are moved backward to an earlier compartment by retrograde transport.moved backward to an earlier compartment by retrograde transport.
In the vesicular transport model, the Golgi cisternae are static In the vesicular transport model, the Golgi cisternae are static organelles, which contain their resident proteins. The passing of organelles, which contain their resident proteins. The passing of molecules from molecules from cis cis to to transtrans through anterograde transport. through anterograde 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 Trans-
Golgi• Retention After
Secretion• Sorting After
Endocytosis• Sorting Signals
Basolateral:Tyrosine orDiLeucine
Apical:N or O-linkedGlycosylationOr 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 Processing of N-linked glycosylation in Golgi
N-linked glycosylation in the Golgi:N-linked glycosylation in the Golgi: > In > In ciscis-Golgi, three mannose residues -Golgi, three mannose residues are removed (1).are removed (1). > In > In medial-medial-Golgi, three GlcNAc (2,4) Golgi, three GlcNAc (2,4) and one fucose (5) residues are and one fucose (5) residues are added, added, while two mannose (3) residues are while two mannose (3) residues are removed.removed. > In > In transtrans-Golgi, three galactose (6) -Golgi, three galactose (6) residues are added, followed by the residues are added, followed by the linkage of N-Acetylneuraminic acid (7) linkage of N-Acetylneuraminic acid (7) on each galactose residue. on each galactose residue.
The Golgi complex is organized into The Golgi complex is organized into 3-4 cisternae, which contain different 3-4 cisternae, which contain different enzymes for protein glycosylation. enzymes for protein glycosylation.
Each enzyme move dynamically from Each enzyme move dynamically from the later to the earlier cisterna through the later to the earlier cisterna through retrograde vesicle transports.retrograde vesicle transports.
(GlcNAc)(GlcNAc)
Evidence of Golgi cisternal maturationEvidence of Golgi cisternal maturation
Yeast cells expressing:Yeast cells expressing: > the > the ciscis-Golgi protein Vrg4-GFP (-Golgi protein Vrg4-GFP (greengreen)) > the > the transtrans-Golgi protein Sec7-DsRed (-Golgi protein Sec7-DsRed (redred) )
A compartment rarely contains both A compartment rarely contains both ciscis- and - and transtrans-Golgi -Golgi proteins at the same time.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 B-subunit (CTXB) uptake to the Golgi complex (marked by -COP)
• Suggests CTXB is delivered to the Golgi complex by a clathrin-independent pathway
-COP: Marker for the Golgi complex
Does internalized CTXB pass through early
endosomes?
• 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
Nichols et al. 2001 J. Cell Biol.
Active Membrane Transport – Review
Process Energy Source Example
Active transport of solutes
ATPMovement of ions across
membranes
Exocytosis ATPNeurotransmitter
secretion
Endocytosis ATPWhite blood cell
phagocytosis
Fluid-phase endocytosis ATPAbsorption by intestinal
cells
Receptor-mediated endocytosis
ATPHormone and cholesterol
uptake
Endocytosis via caveoli ATP Cholesterol regulation
Endocytosis via coatomer vesicles
ATPIntracellular 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.
lysosomeslysosomes Lysosomes contain about Lysosomes contain about 40 types of hydrolytic enzymes40 types of hydrolytic enzymes. For optimal . For optimal activity, they need to be activated by proteolytic cleavage and an acidic activity, they need to be activated by proteolytic cleavage and an acidic environment, which is established by the V-class H+ pumps on lysosomal environment, which is established by the V-class H+ pumps on lysosomal membrane.membrane.
Mature endosomes containing numerous vesicles in their interior are Mature endosomes containing numerous vesicles in their interior are usually called multivesicular endosomes. Fusion of a multivesicular usually called multivesicular endosomes. Fusion of a multivesicular endosome directly with a lysosome releases the internal vesicles into endosome directly with a lysosome releases the internal vesicles into the lumen of the lysosome, where they can be degraded.the lumen of the lysosome, where they can be degraded.
Lysosomal membrane proteins are not incorporated into internal Lysosomal membrane proteins are not incorporated into internal endosomal vesicles, thus keeping them away from degradation.endosomal vesicles, thus keeping them away from degradation.
Formation of multivesicular endosomesFormation of multivesicular endosomes Proteins destined to the multivesicular endosome are tagged with Proteins destined to the multivesicular endosome are tagged with ubiquitin at the plasma membrane, the TGN or the endosomal membrane.ubiquitin at the plasma membrane, the TGN or the endosomal membrane.
In the endosomal budding, a ubiquitin-tagged Hrs protein on the In the endosomal budding, a ubiquitin-tagged Hrs protein on the endosomal membrane facilitates loading of ubiquitinated cargo proteins endosomal membrane facilitates loading of ubiquitinated cargo proteins into vesicle buds and then recruits cytosolic ESCRT proteins to the into vesicle buds and then recruits cytosolic ESCRT proteins to the membrane (step 1).membrane (step 1).
The membrane-associated ESCRT proteins act to complete vesicle The membrane-associated ESCRT proteins act to complete vesicle budding, leading to budding, leading to release of a vesicle release of a vesicle carrying cargo into the carrying cargo into the endosome (step 2).endosome (step 2).
ESCRT proteins are ESCRT proteins are disassembled by the disassembled by the ATPase Vps4 and ATPase Vps4 and returned to the cytosol returned to the cytosol (step 3).(step 3).
Apical-basolateral protein sortingApical-basolateral protein sorting Proteins Proteins destined for either the apical or the basolateral membranesdestined for either the apical or the basolateral membranes are are sorted in the TGN into sorted in the TGN into different transport vesiclesdifferent transport vesicles..
When cells are infected with VSV and influenza viruses simultaneously, When cells are infected with VSV and influenza viruses simultaneously, the VSV G glycoprotein is found only on the basolateral membrane, the VSV G glycoprotein is found only on the basolateral membrane, whereas the influenza HA glycoprotein is found only on the apical whereas the influenza HA glycoprotein is found only on the apical membrane.membrane.
In hepatocytes, membrane In hepatocytes, membrane proteins proteins are directed first to the are directed first to the basolateral basolateral membrane.membrane. Then, both apical and Then, both apical and basolateral proteins are basolateral proteins are endocytosed in the same vesicles:endocytosed in the same vesicles: > the basolateral proteins are > the basolateral proteins are recycled back to basolateral recycled back to basolateral membrane.membrane. > the apical proteins are > the apical proteins are transported across the transported across the cell to apical membrane cell to apical membrane ((transcytosistranscytosis). ).
Intracellular Vesicular Transport
Vesicular transport of neurotransmittersVesicular 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 ImpulsesA. K+ gradients maintained by the Na+-K+ ATPase are responsible for
the resting 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.
B. The action potential: The changes in ion channels and membrane potential.
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 vesiclesCycling of neurotransmitters and synaptic vesicles
Cycling of neurotransmitters and synaptic vesiclesCycling of neurotransmitters and synaptic vesicles
The uncoated vesicles employ a variety of antiporters (blue) to import The uncoated vesicles employ a variety of antiporters (blue) to import neurotransmitters (transmitters) from cytosol (step 1).neurotransmitters (transmitters) from cytosol (step 1).
Transmitter-loaded vesicles move to the active zone (step 2). Transmitter-loaded vesicles move to the active zone (step 2).
Vesicle docks on the membrane of a presynaptic cells, which is Vesicle docks on the membrane of a presynaptic cells, which is mediated mediated by SNAREs. Synaptotagmin, a Ca+2 sensor for exocytosis of transmitter, by SNAREs. Synaptotagmin, a Ca+2 sensor for exocytosis of transmitter, prevents membrane fusion (step 3).prevents membrane fusion (step 3). In response to an In response to an action potentialaction potential, voltage-gated Ca+2 channels in , voltage-gated Ca+2 channels in membrane open, allowing an influx of Ca+2 from the synaptic cleft. It membrane open, allowing an influx of Ca+2 from the synaptic cleft. It causes a conformational change in synaptotagmin, leading to fusion of causes a conformational change in synaptotagmin, leading to fusion of docked vesicles with plasma membrane and release of transmitters into docked vesicles with plasma membrane and release of transmitters into the synaptic cleft (step 4). the synaptic cleft (step 4).
After clathrin/AP vesicles containing v-SNARE and transmitter transporter After clathrin/AP vesicles containing v-SNARE and transmitter transporter proteins bud inward and are pinched off in a dynamin-mediated process, proteins bud inward and are pinched off in a dynamin-mediated process, they loss their coat proteins. At the same time, Na+-transmitter symporters they loss their coat proteins. At the same time, Na+-transmitter symporters take up transmitter from the synaptic cleft (step 5).take up transmitter from the synaptic cleft (step 5).
Vesicles are recovered by endocytosis, creating uncoated vesicles (step Vesicles are recovered by endocytosis, creating uncoated vesicles (step 6).6).
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.
25.6 Budding and fusion reactions
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 endocytosisExocytosis and endocytosis
Exocytosis: a process that a cell releases intracellular molecules Exocytosis: a process that a cell releases intracellular molecules (such as hormones, secretory proteins) contained within a (such as hormones, secretory proteins) contained within a membrane-bounded vesicle by fusion of the vesicle with its plasma membrane-bounded vesicle by fusion of the vesicle with its plasma membrane.membrane.
Endocytosis: a process that a cell uptake extracellular material by Endocytosis: a process that a cell uptake extracellular material by engulfing it within cell, including receptor-mediated engulfing it within cell, including receptor-mediated endocytosis, endocytosis, phagocytosis and pinocytosis.phagocytosis and pinocytosis.
Vesicular transport: transport vesicles carrying material as cargo bud Vesicular transport: transport vesicles carrying material as cargo bud off from the donor compartment and fuse with the target off from the donor compartment and fuse with the target compartment.compartment.
Vesicular Transport: Exocytosis
• Secreting material or replacement of plasma membrane
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).
Introduction
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
Transitional ER site
COPII vesicles
COPI vesiclesRecycling vesicles
To endosomes
To secretory granules
To plasma membrane
cis medial transTGN
TGN = trans-Golgi network
5-48
Regulated secretion
• Occurs in endocrine, exocrine and neuronal cells– Insulin secretion in
pancreatic -cells– Trypsinogen secretion
in pancreatic acinar cells
• Exocytosis occurs in response to a trigger (ex. Ca2+)
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
17-41
Insulin in the regulated secretory pathway
Antibody binds proinsulin (not insulin)
Antibody binds insulin (not proinsulin)
Immature secretory vesicles
Golgi complex
Mature secretory vesicles
Vesicle budding from TGN
Golgi complex
Mature secretory vesicles
Immature secretory vesicles Vesicle
budding from TGN
Clathrin coat
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-GL-GPI with the plasma membrane observed in living cells using total
internal reflection microscopy
Keller et al (2001) Nature Cell Biol.
Mostov et al. 1999 Cell 99:121-122
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
Lodish et al. Figure 17-43
“ Direct” versus “indirect” (transcytotic) trafficking in polarized cells
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• 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– 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
Tuma and Hubbard (2003)
pIgA
pIgA-R
synthesizes IgA
lumen
Blood/interstitial
Ras trafficking via the secretory pathway
Magee and Marshall (1999) Cell
• 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
HRas KRas
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
Lysosomes are small bodies, enclosed
by membranes, that contain hydrolytic
enzymes in eukaryotic cells.
25.7 Protein localization depends on further signals
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?
Kirchhausen 2000 Nature Reviews Molecular Cell Biology 1:187
Key steps in the formation of clathrin-coated vesicles
Activation (TGN)
Activation (PM) Cargo capture
Coat assembly Scission Uncoating
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 cells- exocyst
Lodish et al. Figure 17-51
Sorting signals in cargo molecules
Signal sequence
Type of protein
Transport step
Vesicle type Signal receptor
Mannose-6-phosphate
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
• 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
3D EM tomography of the Golgi complex
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
Interlinked network
Cisternal maturation
Vesicular transport
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?
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.
25.10 Summary
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
