Arf

Golgi to lysosome, melanosome, or platelet vesicles

Each type of AP complex consists of four different subunits. It is not

known whether the coat of AP3 vesicles contains clathrin.

membrane that is typical of a transport vesicle about 50 nm in diameter. Electron micrographs of in vitro budding reactions often reveal structures that exhibit discrete regions of the parent membrane bearing a dense coat accompanied by the curvature characteristic of a completed vesicle (Figure 17-8). Such structures, usually called vesicle buds, appear to be intermediates that are visible after the coat has begun to polymerize but before the completed vesicle pinches off from the parent membrane. The polymerized coat proteins are thought to form some type of curved lattice that drives the formation of a vesicle bud by adhering to the cytosolic face of the membrane.

A Conserved Set of GTPase Switch Proteins Controls Assembly of Different Vesicle Coats

Based on in vitro vesicle-budding reactions with isolated membranes and purified coat proteins, scientists have determined the minimum set of coat components required to form each of the three major types of vesicles. Although most of the coat proteins differ considerably from one type of vesicle to another, the coats of all three vesicles contain a small GTP-binding protein that acts as a regulatory subunit to control coat assembly (see Figure 17-7a). For both COPI and clathrin vesicles, this GTP-binding protein is known as ARF. A different but related GTP-binding protein known as ,Sar1 is present in the coat of COPII vesicles. Both ARF and Sari are monomeric proteins with an overall structure similar to that of Ras, a key intracellular signal-transducing protein (see Figure 14-20). ARF and Sari proteins, like Ras, belong to the GTPase superfamily of switch proteins that cycle between inactive GDP-bound and active GTP-bound forms (see Figure 3-29).

The cycle of GTP binding and hydrolysis by ARF and Sar1 are thought to control the initiation of coat assembly as schematically depicted for the assembly of COPII vesicles in Figure 17-9. First, an ER membrane protein known as Sec12 catalyzes release of GDP from cytosolic Sari • GDP and binding of GTP. The Sec12 guanine nucleotide-exchange factor apparently receives and integrates multiple, as yet unknown signals, probably including the presence of cargo proteins in the ER membrane that are ready to be transported. Binding of GTP causes a conformational change in Sar1 that exposes its hydrophobic N-terminus, which then becomes embedded in the phospholipid bilayer and tethers Sar1 • GTP to the ER membrane. The membrane-attached Sari • GTP drives polymerization of cytosolic complexes of COPII sub-units on the membrane, eventually leading to formation of vesicle buds. Once COPII vesicles are released from the donor membrane, the Sar1 GTPase activity hydrolyzes Sar1 • GTP in the vesicle membrane to Sar1 • GDP with the assistance of one of the coat subunits. This hydrolysis triggers disassembly of the COPII coat. Thus Sari couples a cycle of GTP binding and hydrolysis to the formation and then dissociation of the COPII coat.

ARF protein undergoes a similar cycle of nucleotide exchange and hydrolysis coupled to the assembly of vesicle coats composed either of COPI or of clathrin and other coat proteins (AP complexes) discussed later. A myristate anchor covalently attached to the N-terminus of ARF protein weakly tethers ARF • GDP to the Golgi membrane. When GTP is exchanged for the bound GDP by a nucleotide-exchange factor attached to the Golgi membrane, the resulting conformational change in ARF allows hydrophobic residues in its N-terminal segment to insert into the membrane bilayer. The resulting tight association of ARF • GTP with the membrane serves as the foundation for further coat assembly.

Drawing on the structural similarities of Sari and ARF to other small GTPase switch proteins, researchers have

▲ FIGURE 17-9 Model for the role of Sar1 in the assembly and disassembly of COPII coats. Step 1: Interaction of soluble GDP-bound Sari with the exchange factor Sec12, an ER integral membrane protein, catalyzes exchange of GTP for GDP on Sari. In the GTP-bound form of Sari, its hydrophobic N-terminus extends outward from the protein's surface and anchors Sari to the ER membrane. Step 2|: Sari attached to the membrane serves as a binding site for the the Sec23/Sec24 coat protein complex. Cargo proteins are recruited to the forming vesicle bud by binding of specific short sequences (sorting signals) in their cytosolic regions to sites on the Sec23/Sec24 complex. The coat is completed by assembly of a second type of coat complex composed of Seci3/and Sec3i (not shown). Step 3: After the vesicle coat is complete, the Sec23 coat subunit promotes GTP hydrolysis by Sari. Step 4|: Release of Sari ■ GDP from the vesicle membrane causes disassembly of the coat. [See S. Springer et al., i999, Cell 97:i45.]

▲ EXPERIMENTAL FIGURE 17-10 Coated vesicles accumulate during in vitro budding reactions in the presence of a nonhydrolyzable analog of GTP When isolated Golgi membranes are incubated with a cytosolic extract containing COPI coat proteins and ATP vesicles form and bud off from the membranes. Inclusion of a nonhydrolyzable analog of GTP in the budding reaction prevents disassembly of the coat after vesicle release. This micrograph shows COPI vesicles generated in such a reaction and separated from membranes by centrifugation. Coated vesicles prepared in this way can be analyzed to determine their components and properties. [Courtesy of L. Orci.]

constructed genes encoding mutant versions of the two proteins that have predictable effects on vesicular traffic when transfected into cultured cells. For example, in cells expressing mutant versions of Sari or ARF that cannot hydrolyze GTP, vesicle coats form and vesicle buds pinch off. However, because the mutant proteins cannot trigger disassembly of the coat, all available coat subunits eventually become permanently assembled into coated vesicles that are unable to fuse with target membranes. Addition of a nonhydrolyzable GTP analog to in vitro vesicle-budding reactions causes a similar blocking of coat disassembly. The vesicles that form in such reactions have coats that never dissociate, allowing their composition and structure to be more readily analyzed. The purified COPI vesicles shown in Figure 17-10 were produced in such a budding reaction.

Targeting Sequences on Cargo Proteins Make Specific Molecular Contacts with Coat Proteins

In order for transport vesicles to move specific proteins from one compartment to the next, vesicle buds must be able to discriminate among potential membrane and soluble cargo proteins, accepting only those cargo proteins that should advance to the next compartment and excluding those that should remain as residents in the donor compartment. In addition to sculpting the curvature of a donor membrane, the vesicle coat also functions in selecting specific proteins as cargo. The primary mechanism by which the vesicle coat selects cargo molecules is by directly binding to specific

TABLE 17-2 Known Sorting Signals That Direct Proteins to Specific Transport Vesicles

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