C OATED V ESICLE A SSEMBLY

All types of coated vesicles are formed by polymerization of cytosolic coat proteins on a donor (parent) membrane to form vesicle buds that eventually pinch off from the membrane to release a complete vesicle. Three types of coated vesicles have been characterized, each with a different type of protein coat and each formed by reversible polymerization of a distinct set of GTP-binding proteins (ARF or Sar1) belonging to the GTPase superfamily. Also, each of them transports cargo proteins from particular parent organelles to particular destination organelles:

• COPII vesicles transport proteins from the rough ER to the Golgi.

• COPI vesicles mainly transport proteins in the retrograde direction between Golgi cisternae and from the cis-Golgi back to the rough ER.

• Clathrin vesicles transport proteins from the plasma membrane (cell surface) and the trans-Golgi network to late endosomes.

After formation of vesicles by budding from a donor membrane, the coats depolymerize into their subunits, which are reused to form additional transport vesicles, this is summarized in (Figure 12).

Human Disease Defective cells Defective gene Affected complex Mouse mutant

Hermansky-Pudlak-Syndrome Melanocytes and platelets HPS1 BLOC-3 "Pale ear"

Hermansky-Pudlak-Syndrome Immunocytes, melanocytes & platelets AP3B1 (HPS2) AP-3 "Pearl"

Hermansky-Pudlak-Syndrome Melanocytes and platelets HPS3 BLOC-2 "Cocoa"

Hermansky-Pudlak-Syndrome Melanocytes and platelets HPS4 BLOC-3 "Light ear"

Hermansky-Pudlak-Syndrome Melanocytes and platelets HPS5 BLOC-2 "Ruby-Eye2"

Hermansky-Pudlak-Syndrome Melanocytes and platelets HPS6 BLOC-2 "Ruby-Eye"

Hermansky-Pudlak-Syndrome Melanocytes and platelets DTNBP1 (HPS7) BLOC-1 "Sandy"

Hermansky-Pudlak-Syndrome Melanocytes and platelets AP3D1 AP-3 "Mocha"

Hermansky-Pudlak-Syndrome Immunocytes, melanocytes & platelets RABGGTA Transient with Rabs "Gunmetal"

Hermansky-Pudlak-Syndrome Melanocytes and platelets PLDN BLOC-1 "Pallid"

Hermansky-Pudlak-Syndrome Melanocytes and platelets MU BLOC-1 "Muted"

Hermansky-Pudlak-Syndrome Melanocytes and platelets CNO BLOC-1 "Cappuccino"

Hermansky-Pudlak-Syndrome Melanocytes and platelets VPS33A Unknown "Buff"

Chediak-Higashi-Syndrome Immunocytes and melanocytes LYST Unknown "Beige"

Elejalde-Syndrome (Griscelli 1) Melanocytes and neurons MYO V Rab27a/MyoV/Melanophilin "Dilute"

Griscelli-Syndrome 2 Immunocytes and melanocytes RAB27A Rab27a/MyoV/Melanophilin "Ashen"

Griscelli-Syndrome 3 Melanocytes MLPH Rab27a/MyoV/Melanophilin "Leaden"

Human Disease Defective cells Defective gene Affected complex Mouse mutant

Hermansky-Pudlak-Syndrome Melanocytes and platelets HPS1 BLOC-3 "Pale ear"

Hermansky-Pudlak-Syndrome Immunocytes, melanocytes & platelets AP3B1 (HPS2) AP-3 "Pearl"

Hermansky-Pudlak-Syndrome Melanocytes and platelets HPS3 BLOC-2 "Cocoa"

Hermansky-Pudlak-Syndrome Melanocytes and platelets HPS4 BLOC-3 "Light ear"

Hermansky-Pudlak-Syndrome Melanocytes and platelets HPS5 BLOC-2 "Ruby-Eye2"

Hermansky-Pudlak-Syndrome Melanocytes and platelets HPS6 BLOC-2 "Ruby-Eye"

Hermansky-Pudlak-Syndrome Melanocytes and platelets DTNBP1 (HPS7) BLOC-1 "Sandy"

Hermansky-Pudlak-Syndrome Melanocytes and platelets AP3D1 AP-3 "Mocha"

Hermansky-Pudlak-Syndrome Immunocytes, melanocytes & platelets RABGGTA Transient with Rabs "Gunmetal"

Hermansky-Pudlak-Syndrome Melanocytes and platelets PLDN BLOC-1 "Pallid"

Hermansky-Pudlak-Syndrome Melanocytes and platelets MU BLOC-1 "Muted"

Hermansky-Pudlak-Syndrome Melanocytes and platelets CNO BLOC-1 "Cappuccino"

Hermansky-Pudlak-Syndrome Melanocytes and platelets VPS33A Unknown "Buff"

Chediak-Higashi-Syndrome Immunocytes and melanocytes LYST Unknown "Beige"

Elejalde-Syndrome (Griscelli 1) Melanocytes and neurons MYO V Rab27a/MyoV/Melanophilin "Dilute"

Griscelli-Syndrome 2 Immunocytes and melanocytes RAB27A Rab27a/MyoV/Melanophilin "Ashen"

Griscelli-Syndrome 3 Melanocytes MLPH Rab27a/MyoV/Melanophilin "Leaden"

Table 3: Mouse models of lipid trafficking disorders

The general scheme of vesicle budding shown in Figure 13 applies to all three known types of coated vesicles.

Figure 12: Involvement of the three major types of coat proteins in vesicular traffic in the secretory and endocytic pathways.

A. 1.COPII vesicles mediate anterograde transport from the rough ER to the cis-Golgi/cis-Golgi network. 2. COPI vesicles mediate retrograde transport within the Golgi and from the cis-Golgi/cis-Golgi network to the rough ER. 3.

The coat proteins surrounding secretory vesicles are not yet characterized;

these vesicles carry secreted proteins and plasma-membrane proteins from the trans-Golgi network to the cell surface. 4-5. Vesicles coated with clathrin bud from the trans-Golgi network and from the plasma membrane; after uncoating, these vesicles fuse with late endosomes. Adapted from Alberts 2003, pg. 719

Lipid droplet

UC + SPM ABCA1 pathway

NPC

Figure 13: Budding and fusion. Overview of vesicle budding and fusion with a target membrane. (a) Budding is initiated by recruitment of a small GTP-binding protein to a patch of donor membrane. Complexes of coat proteins in the cytosol then bind to the cytosolic domain of membrane cargo proteins, some of which also act as receptors that bind soluble proteins in the lumen, thereby recruiting luminal cargo proteins into the budding vesicle. (b) After being released and shedding its coat, a vesicle fuses with its target membrane in a process that involves interaction of cognate T-SNARE proteins [Alberts, 2003].

B. Vesicle fusion A. Vesicle budding

Shortly after a vesicle buds off from the donor membrane, the vesicle coat disassembles to uncover a vesicle-specific membrane protein, a v-SNARE. Likewise, each type of target membrane in a cell contains t-SNARE membrane proteins. After Rab-mediated docking of a vesicle on its target (destination) membrane, the interaction of cognate SNAREs brings the two membranes close enough together that they can fuse. The SNARE-mediated vesicle fusion can be viewed in figure 14.

Figure 14: Syntaxins and SNAREs in vesicle fusion. A v-SNARE, known as VAMP (vesicle-associated membrane protein), is incorporated into secretory vesicles as they bud from the trans-Golgi network. Syntaxins are t-SNAREs, integral membrane protein in the plasma membrane, and SNAP-25, which is attached to the plasma membrane by a hydrophobic lipid anchor in the middle of the protein. The cytosolic region in each of these three SNARE proteins contains a repeating sequence that allows four α−helices - one from VAMP, one from syntaxin, and two from SNAP-25 to coil around one another to form a four-helix bundle. As the four-helix bundles form, the vesicle and target membranes are drawn together by the embedded transmembrane domains of VAMP and syntaxin, an effect induced by Ca⁺⁺ ions.