MCB 380 Lecture Outline - Spring 2007

Lecture 9 (Molecular Mechanisms of Vesicular Traffic, Section 17.2)

10. Figure 17-50, Lodish 4e. Involvement of the three known types of coat proteins- COP I, COP II and clathrin - in vesicular traffic in the secretory and endocytic pathways. Clathrin coats vesicles destined for the endosomes. The origins of these vesicles can be the plasma membrane and the trans-Golgi network. The difference in these vesicles is the composition of the assembly particle, which is AP1 if the Golgi is the origin and it is AP2 when the plasma membrane is the origin. AP1 and AP2 complexes contain different adapter proteins. COPI coated vesicles participate in the retrograde transport pathway between Golgi cristae and in trafficking from the cis-Golgi back to the ER. COPII coated vesicles bud from the rough ER and some move directly to the cis-Golgi whereas others fuse with other vesicles to produce an ER-Golgi intermediate compartment. All of these COPII coated vesicles move along microtubules and are affected by drugs that either block the assembly or cause disassembly of microtubules.

11. Figure 17-7, Lodish5e: Overview of vesicle budding from donor membranes (panel A) and fusion with a target membrane (panel B) .

Panel A: This image shows the features common to all three forms of vesicle budding: a) small GTP-binding proteins that are recruited to a patch of donor membrane to initiate budding and that regulate the rate of vesicle formation, b) adapter proteins that link a particular cargo protein (either a membrane cargo protein directly or a soluble protein indirectly via a membrane cargo receptor protein) to a particular type of coat, c) coat protein subunits whose reversible polymerization around the cytosolic face of a forming vesicle help to drive the budding process. The polymerizing coat proteins add curvature to the membrane during budding and also participate in the selection of the cargo to be carried by the vesicle through interactions with the adapter proteins.

Panel B: Before a coated vesicle can fuse with its target or destination membrane, it must shed its coat. This is because fusion requires direct interactions of the membrane lipids of the vesicles and target membranes. The actual fusion process involves the interaction of proteins called SNAREs that have unusual protein structural features. Some of these interaction proteins are in the vesicle membrane and are appropriately called vSNAREs. They interact with tSNAREs in the target membrane to pull the membranes close enough together to fuse the two lipid bilayers.

12. Table 17-1, Lodish 5e: Coated vesicles involved in protein trafficking. This table provides a useful summary of the three known types of coated vesicles, which are named for the primary coat protein each carries. The transport-mediated step is shown for each type of vesicle. Note that it is not known what type of vesicle moves between the trans-Golgi network and the plasma membrane.

13. Figure 17-8, Lodish 5e: Vesicle buds can be visualized during in vitro budding reactions. Here is the result of incubating in vitro purified COPII coat proteins with isolated ER vesicles or with artificial phospholipid vesicles called liposomes. The coat proteins polymerize on the surface of the vesicles and cause the protrusion of highly curved buds. The vesicle coat is visible as a dark, electron dense layer on the vesicle buds in this electron micrograph.

14. Figure 17-9, Lodish 5e: Role of Sar1 in the assembly and disassembly of COPII coats. A big part of the control of vesicle coats falls to an evolutionarily conserved set of GTPase switch proteins (members of the GTPase superfamily). Both ARF and Sar1 are related to Ras, a protein involved in cell signaling. ARF controls the coat assembly of COPI vesicles and clathrin-coated vesicles whereas Sar1 controls the coat assembly of COPII vesicles, as shown in this model. The GDP-bound form of Sar1, Sar1(GDP) interacts with an integral membrane protein of the ER called Sec12, which is a GDP/GTP exchange factor. Sar1(GTP) undergoes a conformational change, extending a hydrophobic N-terminal polypeptide segment that anchors it to the ER membrane. Attached Sar1(GTP) provides a binding site for coat protein complex Sec23/Sec24. Intregral membrane cargo proteins are recruited by their amino acid sequences called sorting signals that bind directly to coat protein complexes on the cytosolic face of the budding vesicles. Other cargo proteins in the lumen of the ER for example may bind to these membrane cargo proteins. Additional coat complex Sec13/Sec31 is added to complete the coat assembly. The Sec23 coat protein interacts with Sar1(GTP) stimulating hydrolysis of GTP by Sar1. The Sar1(GDP) is released causing disassembly of the coat.

15. Figure 13-8, Lodish 5e: How the switches in GTPase proteins work. Note the two switch regions shown in blue (switch II) and green (switch I). The G protein is in the on or active state with GTP bound as shown in panel a. These switches interact with the gamma phosphate of GTP through highly conserved threonine and glycine residues. Hydrolysis and release of the gamma phosphate by the hydrolytic activity that is part of the G protein (stimulated by GTPase accelerating protein GAP) produces GDP, breaks these bonds with the amino acids, and allows the G protein to relax into a second shape or conformation called the off or inactive state (panel b). This is shown in this diagram for Ras, a monomeric G protein. When GTP is bound in exchange for GDP, faciliated by a guanine nucleotide-exchange factor (GEF), the cycle is completed and ready for another round.

16. Figure 17-10, Lodish 5e: Coated vesicles accumulate during in vitro budding reactions in the presence of nonhydrolyzable GTP analog. Electron micrograph of COP I vesicles purified by centrifugation to separate them from the Golgi fraction of rat hepatocytes after incubation with cytosol and ATP to stimulate vesicle formation. The coat of these vesicles is formed from coatomers which consist of seven polypeptide subunits. Some of these subunits function like the assembly particles of clathrin-coated vesicles. The addition of a nonhydrolyzable analog of GTP to the budding reaction blocks the disassembly of the coat that would normally occur after vesicle release. The coated vesicles are essentially trapped in this way.

17. Table 17-2, Lodish 5e: Known sorting signals that direct proteins to specific transport vesicles. This is a good summary table. The key point is that these sorting signals are amino acid sequences in the cytosolic domains of transmembrane cargo proteins. The polymerized coat functions as a binding matrix to cluster bound membrane proteins that are selected based on their sorting signals into budding vesicles. These transmembrane cargo proteins can also act as cargo receptors, interacting by their lumenal domains with other cargo proteins. This is how the cargo is loaded into the correct molecular truck or vesicle.

18. Figure 17-11, Lodish 5e: Model for docking and fusion of transport vesicles with their target membranes. The small G protein family known as Rab functions as a molecular switch, like Sar1 and ARF. Cycles of GTP binding and hydrolysis accompanied by conformational changes that drive the insertion of Rab's hydrophobic membrane anchor and allow binding to downstream effector proteins are involved. Rab proteins in the vesicle may be 'timers' for targeting and fusion. Key to transport vesicle-target membrane interaction is the formation of a coiled coil structure or four-helix bundle composed of four long helices containing T- and V-SNARE proteins (1 each) and SNAP25 (2 helices). T stands for target membrane and V stands for vesicle membrane. The interaction of these so-called cognate SNAREs pulls the two membranes close enough together to fuse. Now it is clear why vesicles must lose their coats before membrane fusion can occur. A pre-fusion complex forms which probably consists of a number of coiled coil complexes. In this figure SNARE-mediated fusion during protein secretion is used as an example. The SNARE called VAMP is incorporated into vesicles during budding at the trans-Golgi network. After membrane fusion, the SNAREs undergo a major conformational change to form the cis-SNARE complexes. Now the SNAREs have done their work and must be dissociated to allow them to be reused. Two proteins, NSF (N-ethylmaleimide-sensitive factor), and alpha-SNAP (soluble NSF attachment protein) are involved. They both bind to a SNARE complex and then bound NSF hydrolyzes ATP. The energy of hydrolysis is used to dissociate the SNARE complex, the components then sit in the target membrane awaiting reuse. Note: Figure 3-6, Lodish 5e: Molecular structure of heptad repeat coil.

19. Figure 17-12, Lodish 5e: Schematic models of the structure of influenza hemagglutinin (HA) at pH 7 and 5. Influenza virus has been a model not only for glycoprotein biosynthesis and protein trafficking in cells but also a model for membrane fusion. Flu virions have an envelope derived from the plasma membrane of infected cells. These virions are taken up by cells by endocytosis after the virions have bound to the cell surface at pH 7 by glycoprotein spikes like HA. The viral core or nucleocapsid must still get out of the endosomal vesicle and enter the cytosol in order to successfully infect the cell. To do this it uses HA to fuse the endosomal vesicle's membrane with its own viral envelope, allowing the viral nucleocapsid carrying the viral genes to enter the cytosol. The acidic pH of the endosomal compartment is thought to trigger a dramatic conformational change in the HA spikes, exposing highly hydrophobic fusion peptides that poke into the endosomal membrane and stimulate fusion of each leaflet with the viral envelope.

20. Figure 17-13, Lodish 5e: This shows how the fusion may occur. This may also be a model for SNARE mediated fusion, where HA plays the role of the SNAREs. A scaffold first forms composed of multiple copies of HA which brings the two membranes close together, as SNARES do as well. Then, the exoplasmic leaflets fuse, followed by the cytosolic leaflets, and this forms a pore that increases in size until the two membranes are completely joined.

Return to lecture index page