Dynamin is a 100-kDa GTPase that assembles into multimeric spirals at the necks of budding clathrin-coated vesicles. We describe three different intramolecular binding interactions that may account for the process of dynamin self-assembly. The first binding interaction is the dimerization of a 100-amino acid segment in the C-terminal half of dynamin. We call this segment the assembly domain, because it appears to be critical for multimerization.

Clathrin-coated vesicles mediate diverse processes such as nutrient uptake, downregulation of hormone receptors, formation of synaptic vesicles, virus entry, and transport of biosynthetic proteins to lysosomes. Cycles of coat assembly and disassembly are integral features of clathrin-mediated vesicular transport (Fig. 1a). Coat assembly involves recruitment of clathrin triskelia, adaptor complexes and other factors that influence coat assembly, cargo sequestration, membrane invagination and scission (Fig. 1a). Coat disassembly is thought to be essential for fusion of vesicles with target membranes and for recycling components of clathrin coats to the cytoplasm for further rounds of vesicle formation. In vitro, cytosolic heat-shock protein 70 (Hsp70) and the J-domain co-chaperone auxilin catalyse coat disassembly. However, a specific function of these factors in uncoating in vivo has not been demonstrated, leaving the physiological mechanism and significance of uncoating unclear. Here we report the identification and characterization of a Saccharomyces cerevisiae J-domain protein, Aux1. Inactivation of Aux1 results in accumulation of clathrin-coated vesicles, impaired cargo delivery, and an increased ratio of vesicle-associated to cytoplasmic clathrin. Our results demonstrate an in vivo uncoating function of a J domain co-chaperone and establish the physiological significance of uncoating in transport mediated by clathrin-coated vesicles.

Clathrin, a multimeric protein involved in intracellular protein trafficking, is composed of three heavy chains (Chc) and three light chains (Clc). Upon disruption (clc1⌬) of the single Clc-encoding gene (CLC1) in yeast, the steady state protein levels of Chc decreased 5-10-fold compared with wild type cells; consequently, phenotypes exhibited by clc1⌬ cells may result indirectly from the loss of Chc as opposed to the absence of Clc. As an approach to directly examine Clc function, clc1⌬ strains were generated that carry a multicopy plasmid containing the clathrin heavy chain gene (CHC1), resulting in levels of Chc 5-10-fold elevated over wild-type levels. As with deletion of CHC1, deletion of CLC1 results in defects in growth, receptor-mediated endocytosis, and maturation of the mating pheromone ␣-factor. However, elevated Chc expression in clc1⌬ cells partially suppresses the growth and ␣-factor maturation defects displayed by clc1⌬ cells alone. Biochemical analyses indicate that trimerization and assembly of Chc are perturbed in the absence of Clc, resulting in vesiculation defects. Our results demonstrate that the light chain subunit of clathrin is required for efficient Chc trimerization, proper formation of clathrin coats, and the generation of clathrin-coated vesicles.Distinct compartments in eukaryotic cells are maintained through the selective transport of proteins carried out by small vesicular carriers. Generation of these vesicles involves assembly of proteinaceous coats on the cytoplasmic surface of donor compartment membranes, which leads to the budding of coated vesicles. Although a variety of proteins are transported through vesicular movement, a subset of specific trafficking events are mediated by clathrin-coated vesicles. These include the retention of resident Golgi membrane proteins, receptormediated endocytosis, and the sorting of lysosomal/vacuolar proteins from the secretory pathway to the lysosome/vacuole (1, 2).The clathrin molecule, or triskelion, is a hexamer composed of three heavy chain subunits (Chc) and three light chain subunits (Clc) (1). In mammals, there are two forms of light chain, LC a and LC b , which share 60% amino acid identity and appear to be randomly distributed in clathrin trimers. In yeast, there is only one form of light chain encoded by the CLC1 gene (3). Formation of a clathrin-coated vesicle is initiated by binding of clathrin-associated protein complexes (APs) 1 to the donor membrane. Triskelions associate with the APs and polymerize into polygonal lattice structures. Such clathrin-coated membrane segments, known as coated pits, are thought to collect specific cargo proteins through interactions between the cargo protein cytoplasmic domains and the AP complexes. The clathrin-coated pit then invaginates and pinches off to form a clathrin-coated vesicle carrying the selected cargo proteins. Formation of the vesicle may involve rearrangement of subunits assembled on the coated pit, or it may be driven by the polymerization of new clathrin subunits into a polyhe...

The distinctive triskelion shape of clathrin allows assembly into polyhedral lattices during the process of clathrin‐coated vesicle formation. We have used random and site‐directed mutagenesis of the yeast clathrin heavy chain gene (CHC1) to characterize regions which determine Chc trimerization and binding to the clathrin light chain (Clc) subunit. Analysis of the mutants indicates that mutations in the trimerization domain at the triskelion vertex, as well as mutations in the adjacent leg domain, frequently influence Clc binding. Strikingly, one mutation in the trimerization domain enhances the association of Clc with Chc. Additional mutations in the trimerization domain, in combination with mutations in the adjacent leg domain, exhibit severe defects in Clc binding while maintaining near normal trimerization properties. The position of these trimerization domain mutations on one face of a putative α‐helix defines a region on the trimer surface that interacts directly with Clc. These results suggest that Clc extends into the Chc trimerization domain from the adjacent leg, thereby bridging the two domains. On the basis of this conclusion, we propose a new model for the organization of the triskelion vertex which provides a structural basis for regulatory effects of Clc on clathrin function.

et al. (1998) report in this issue of Cell the crystal structure of the clathrin heavy chain amino-terminal domain, a site of key interactions within clathrin coats. Clathrin Coat Components Flux of membrane and proteins through secretory and endocytic pathways in eukaryotic cells is mediated by Two oligomeric protein complexes, clathrin and adaptor proteins (APs), are major constituents of clathrin coats transport vesicles. Genesis of these vesicles involves assembly of cytoplasmic coat protein complexes onto (reviewed in Schmid, 1997). Clathrin is a three-legged molecule, formed by C-terminal association of three the donor organelle membrane. Three basic classes of vesicle coats have been identified: clathrin, COPI, and elongated heavy chains (HC), each carrying a light chain (LC) (Figure 1B). The distinctive clathrin shape, a triskel-COPII (reviewed in Schmid, 1997, and references therein). Coats are thought to physically deform a patch of the ion, is well-adapted for assembly into polygonal arrays characteristic of clathrin coats (Figure 1C). In low ionic donor organelle membrane into a new vesicle and sequester selected membrane proteins into the emerging strength, mildly acidic, high Ca 2ϩ buffers, purified clathrin triskelia self-assemble into closed polyhedral cages re-vesicle (Figure 1A). Proteins packaged into coated vesicles include cargo in transit to other organelles and sembling the coats on transport vesicles. Removal of LC subunits from triskelia does not prevent cage formation, machinery to guide and dock each vesicle to the target organelle. Thus, coats have a central role in orchestrat-indicating that self-assembly is an inherent property of HC (Ungewickell and Ungewickell, 1991, and references ing traffic between membrane organelles. Once coated vesicles have formed, coat disassembly allows vesicle therein). However, compared to intact clathrin, LC-free triskelia assemble more readily in the absence of Ca 2ϩ . fusion with the target membrane and returns coat complexes to the cytoplasm for additional cycles of vesicle These properties suggest that LC might inhibit spontaneous clathrin assembly, thereby ensuring assembly formation (Figure 1A). Since their discovery in the 1960s, clathrin coats have served as a paradigm for under-only at membrane sites destined for vesiculation (Ungewickell and Ungewickell, 1991). LC subunits can be standing the molecular basis of vesicle formation (reviewed in Schmid, 1997). Clathrin coats act at the plasma phosphorylated and bind Ca 2ϩ , calmodulin, and hsc70, all potential regulatory components (Figure 1B). membrane to form endocytic transport vesicles, at the trans-Golgi network (TGN) to form endosome-targeted AP complexes act in both clathrin assembly and cargo

The role of clathrin light chain phosphorylation in regulating clathrin function has been examined in Saccharomyces cerevisiae. The phosphorylation state of yeast clathrin light chain (Clc1p) in vivo was monitored by [32P]phosphate labeling and immunoprecipitation. Clc1p was phosphorylated in growing cells and also hyperphosphorylated upon activation of the mating response signal transduction pathway. Mating pheromone-stimulated hyperphosphorylation of Clc1p was dependent on the mating response signal transduction pathway MAP kinase Fus3p. Both basal and stimulated phosphorylation occurred exclusively on serines. Mutagenesis of Clc1p was used to map major phosphorylation sites to serines 52 and 112, but conversion of all 14 serines in Clc1p to alanines [S(all)A] was necessary to eliminate phosphorylation. Cells expressing the S(all)A mutant Clc1p displayed no defects in Clc1p binding to clathrin heavy chain, clathrin trimer stability, sorting of a soluble vacuolar protein, or receptor-mediated endocytosis of mating pheromone. However, the trans-Golgi network membrane protein Kex2p was not optimally localized in mutant cells. Furthermore, pheromone treatment exacerbated the Kex2p localization defect and caused a corresponding defect in Kex2p-mediated maturation of the α-factor precursor. The results reveal a novel requirement for clathrin during the mating response and suggest that phosphorylation of the light chain subunit modulates the activity of clathrin at the trans-Golgi network.