O

(a) Triskelion structure

Heavy Light chain chain

(b) Assembly intermediate

(a) Triskelion structure

Heavy Light chain chain

Binding site for assembly particles

(b) Assembly intermediate

Binding site for assembly particles

▲ FIGURE 17-19 Structure of clathrin coats. (a) A

clathrin molecule, called a triskelion, is composed of three heavy and three light chains. It has an intrinsic curvature due to the bend in the heavy chains. (b) The fibrous clathrin coat around vesicles is constructed of 36 clathrin triskelions. Depicted here is an intermediate in assembly of a clathrin coat, containing 10 of the final 36 triskelions, which illustrates the intrinsic curvature and the packing of clathrin triskelions. (c) Clathrin coats were formed in vitro by mixing purified clathrin heavy and light chains with AP2 complexes in the absence of membranes. Cryoelectron micrographs of more than 1000 assembled particles were analyzed by digital image processing to generate an average structural representation. The left image shows the reconstructed structure of a complete particle with AP2 complexes packed into the interior of the clathrin cage. In the right image, the AP2 complexes have been subtracted to show only the assembled clathrin heavy and light chains. [See B. Pishvaee and G. Payne, 1998, Cell 95:443. Part (c) from Corinne J. Smith, Department of Biological Sciences, University of Warwick.]

(«35,000-40,000 MW). Triskelions polymerize to form a polygonal lattice with an intrinsic curvature (Figure i7-i9b). When clathrin polymerizes on a donor membrane, it does so in association with AP complexes, which assemble between the clathrin lattice and the membrane. Each AP complex (340,000 MW) contains one copy each of four different adapter subunit proteins. A specific association between the globular domain at the end of each clathrin heavy chain in a triskelion and one subunit of the AP complex both promotes the co-assembly of clathrin triskelions with AP complexes and adds to the stability of the completed vesicle coat (Figure i7-i9c).

By binding to the cytosolic face of membrane proteins, adapter proteins determine which cargo proteins are specifically included in (or excluded from) a budding transport vesicle. Each type of AP complex (e.g., APi, AP2, AP3) and the recently identified GGAs are composed of different, though related, proteins. Vesicles containing each complex have been found to mediate specific transport steps (see Table i7-i). All vesicles whose coats contain one of these complexes utilize ARF to initiate coat assembly onto the donor membrane. As discussed previously, ARF also initiates assembly of COPI coats. The additional features of the membrane or protein factors that determine which type of coat will assemble after ARF attachment are not well understood at this time.

Vesicles that bud from the trans-Golgi network en route to the lysosome by way of the late endosome have clathrin coats associated with either APi or GGA. Both APi and GGA bind to the cytosolic domain of cargo proteins in the donor membrane, but the functional differences between vesicles that contain APi or GGA are unclear. Recent studies have shown that membrane proteins containing a Tyr-X-X-$ sequence, where X is any amino acid and $ is a bulky hydrophobic amino acid, are recruited into clathrin/APi vesicles budding from the trans-Golgi network. This [email protected] sorting signal interacts with one of the APi subunits in the vesicle coat. As we discuss in the next section, vesicles with clathrin/AP2 coats, which bud from the plasma membrane during endocytosis, also can recognize the YXX$ sorting signal.

Some vesicles that bud from the trans-Golgi network have coats composed of the AP3 complex. These vesicles mediate trafficking to the lysosome, but they appear to bypass the late endosome and fuse directly with the lysosomal membrane. In certain types of cells, such AP3 vesicles mediate protein transport to specialized storage compartments related to the lysosome. For example, AP3 is required for delivery of proteins to melanosomes, which contain the black pigment melanin in skin cells, and to platelet storage vesicles in megakaryocytes, a large cell that fragments into dozens of platelets. Mice with mutations in either of two different subunits of AP3 not only have abnormal skin pigmentation but also exhibit bleeding disorders. The latter occur because tears in blood vessels cannot be repaired without platelets that contain normal storage vesicles.

Dynamin Is Required for Pinching Off of Clathrin Vesicles

A fundamental step in the formation of a transport vesicle that we have not yet considered is how a vesicle bud is pinched off from the donor membrane. In the case of clathrin/AP-coated vesicles, a cytosolic protein called dynamin is essential for release of complete vesicles. At the later stages of bud formation, dynamin polymerizes around the neck portion and then hydrolyzes GTP. The energy derived from GTP hydrolysis is thought to drive "contraction" of dynamin around the vesicle neck until the vesicle pinches off (Figure 17-20). Interestingly, COPI and COPII vesicles appear to pinch off from donor membranes without the aid of a GTPase such as dynamin. At present this fundamental difference in the process of pinching off among the different types of vesicles is not understood.

Incubation of cell extracts with a nonhydrolyzable derivative of GTP provides dramatic evidence for the importance of dynamin in pinching off of clathrin/AP vesicles during en-docytosis. Such treatment leads to accumulation of clathrin-coated vesicle buds with excessively long necks that are

▲ FIGURE 17-20 Model for dynamin-mediated pinching off of clathrin/AP-coated vesicles. After a vesicle bud forms, dynamin polymerizes over the neck. By a mechanism that is not well understood, dynamin-catalyzed hydrolysis of GTP leads to release of the vesicle from the donor membrane. Note that membrane proteins in the donor membrane are incorporated into vesicles by interacting with AP complexes in the coat. [Adapted from K. Takel et al., i995, Nature 374:i86.]

▲ EXPERIMENTAL FIGURE 17-21 GTP hydrolysis by dynamin is required for pinching off of clathrin-coated vesicles in cell-free extracts. A preparation of nerve terminals, which undergo extensive endocytosis, was lysed by treatment with distilled water and incubated with GTP-y-S, a nonhydrolyzable derivative of GTP After sectioning, the preparation was treated with gold-tagged anti-dynamin antibody and viewed in the electron microscope. This image, which shows a long-necked clathrin/AP-coated bud with polymerized dynamin lining the neck, reveals that buds can form in the absence of GTP hydrolysis, but vesicles cannot pinch off. The extensive polymerization of dynamin that occurs in the presence of with GTP- y-S probably does not occur during the normal budding process. [From K. Takel et al., i995, Nature 374:i86; courtesy of Pietro De Camilli.]

surrounded by polymeric dynamin but do not pinch off (Figure 17-21). Likewise, cells expressing mutant forms of dy-namin that cannot bind GTP do not form clathrin-coated vesicles, and instead accumulate similar long-necked vesicle buds encased with polymerized dynamin.

As with COPI and COPII vesicles, clathrin/AP vesicles normally lose their coat soon after their formation. Cytosolic Hsc70, a constitutive chaperone protein found in all eukaryotic cells, is thought to use energy derived from the hydrolysis of ATP to drive depolymerization of the clathrin coat into triske-lions. Uncoating not only releases triskelions for reuse in the formation of additional vesicles, but also exposes v-SNAREs for use in fusion with target membranes. Conformational changes that occur when ARF switches from the GTP-bound to GDP-bound state are thought to regulate the timing of clathrin coat depolymerization. How the action of Hsc70 might be coupled to ARF switching is not well understood.

Mannose 6-Phosphate Residues Target Soluble Proteins to Lysosomes

Most of the sorting signals that function in vesicular trafficking are short amino acid sequences in the targeted protein. In contrast, the sorting signal that directs soluble

Exoplasmic face

Soluble

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