sequences, or sorting signals, in the cytosolic portion of membrane cargo proteins (see Figure 17-7a). The polymerized coat thus acts as an affinity matrix to cluster selected membrane cargo proteins into forming vesicle buds. Soluble proteins within the lumen of parent organelles can in turn be selected by binding to the luminal domains of certain membrane cargo proteins, which act as receptors for luminal cargo proteins. The properties of several known sorting signals in membrane and soluble proteins are summarized in Table 17-2. We describe the role of these signals in more detail in later sections.

Rab GTPases Control Docking of Vesicles on Target Membranes

A second set of small GTP-binding proteins, known as Rab proteins, participate in the targeting of vesicles to the appropriate target membrane. Like Sari and ARF, Rab proteins belong to the GTPase superfamily of switch proteins. Conversion of cytosolic Rab • GDP to Rab • GTP, catalyzed by a specific guanine nucleotide-exchange factor, induces a conformational change in Rab that enables it to interact with a surface protein on a particular transport vesicle and insert its isoprenoid anchor into the vesicle membrane. Once

Rab • GTP is tethered to the vesicle surface, it is thought to interact with one of a number of different large proteins, known as Rab effectors, attached to the target membrane. Binding of Rab • GTP to a Rab effector docks the vesicle on an appropriate target membrane (Figure 17-11, step 1). After vesicle fusion occurs, the GTP bound to the Rab protein is hydrolyzed to GDP, triggering the release of Rab • GDP, which then can undergo another cycle of GDP-GTP exchange, binding, and hydrolysis.

Several lines of evidence support the involvement of specific Rab proteins in vesicle-fusion events. For instance, the yeast SEC4 gene encodes a Rab protein, and yeast cells expressing mutant Sec4 proteins accumulate secretory vesicles that are unable to fuse with the plasma membrane (class E mutants in Figure 17-5). In mammalian cells, Rab5 protein is localized to endocytic vesicles, also known as early endo-somes. These uncoated vesicles form from clathrin-coated vesicles just after they bud from the plasma membrane during endocytosis (see Figure 17-1, step 9). The fusion of early endosomes with each other in cell-free systems requires the presence of Rab5, and addition of Rab5 and GTP to cell-free extracts accelerates the rate at which these vesicles fuse with each other. A long coiled protein known as EEA1 (early endosome antigen 1), which resides on the membrane of the early endosome, functions as the effector for Rab5. In this case, Rab5 • GTP on one endocytic vesicle is thought to specifically bind to EEA1 on the membrane of another endocytic vesicle, setting the stage for fusion of the two vesicles.

Similarly, Rab1 is essential for ER-to-Golgi transport reactions to occur in cell-free extracts. Rab1 • GTP binds to a long coiled-coil protein known as p115, which specifically tethers COPII vesicles carrying Rab1- GTP to the target Golgi membrane. A different type of Rab effector appears to function for each vesicle type and at each step of the secretory pathway. Many questions remain about how Rab proteins are targeted to the correct membrane and how specific complexes form between the different Rab proteins and their corresponding effector proteins.

Paired Sets of SNARE Proteins Mediate Fusion of Vesicles with Target Membranes

As noted previously, shortly after a vesicle buds off from the donor membrane, the vesicle coat disassembles to uncover a vesicle-specific membrane protein, a v-SNARE (see Figure 17-7b). 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.

One of the best-understood examples of SNARE-mediated fusion occurs during exocytosis of secreted proteins (Figure 17-11, steps 2 and 3). In this case, the v-SNARE, known as VAMP (vesicle-associated membrane protein), is incorporated into secretory vesicles as they bud from the trans-Golgi network. The t-SNAREs are syntaxin, an integral membrane protein in the plasma membrane, and sSNAP-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 hep-tad sequence that allows four a helices—one from VAMP, one

M FIGURE 17-11 Model for docking and fusion of transport vesicles with their target membranes. The proteins shown in this example participate in fusion of secretory vesicles with the plasma membrane, but similar proteins mediate all vesicle-fusion events. Step 1: A Rab protein tethered via a lipid anchor to a secretory vesicle binds to an effector protein complex on the plasma membrane, thereby docking the transport vesicle on the appropriate target membrane. Step 2|: A v-SNARE protein (in this case, VAMP) interacts with the cytosolic domains of the cognate t-SNAREs (in this case, syntaxin and SNAP-25). The very stable coiled-coil SNARE complexes that are formed hold the vesicle close to the target membrane. Inset: Numerous noncovalent interactions between four long a helices, two from SNAP-25 and one each from syntaxin and VAMP stabilize the coiled-coil structure. Step 3: Fusion of the two membranes immediately follows formation of SNARE complexes, but precisely how this occurs is not known. Step 4|: Following membrane fusion, NSF in conjunction with a-SNAP protein binds to the SNARE complexes. The NSF-catalyzed hydrolysis of ATP then drives dissociation of the SNARE complexes, freeing the SNARE proteins for another round of vesicle fusion. [See J. E. Rothman and T. Söllner, 1997, Science 276:1212, and W. Weis and R. Scheller, 1998, Nature 395:328. Inset from Y A. Chen and R. H. Scheller, 2001, Nat. Rev. Mol. Cell Biol. 2(2):98.]

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