Identification of Domain Required for Catalytic Activity of Auxilin in Supporting Clathrin Uncoating by Hsc70

Abstract

During clathrin-mediated endocytosis Hsc70, supported by the J-domain protein auxilin, uncoats clathrin-coated vesicles. Auxilin contains both a clathrinbinding domain and a J-domain that binds Hsc70, and it has been suggested that these two domains are both necessary and sufficient for auxilin activity. To test this hypothesis, we created a chimeric protein consisting of the J-domain of auxilin linked to the clathrin-binding domain of the assembly protein AP180. This chimera supported uncoating, but unlike auxilin it acted stoichiometrically rather than catalytically because, like Hsc70, it remained associated with the uncoated clathrin. This observation supports our proposal that Hsc70 chaperones uncoated clathrin by inducing formation of a stable Hsc70-clathrin-AP complex. It also shows that Hsc70 acts by dissociating individual clathrin triskelions rather than cooperatively destabilizing clathrincoated vesicles. Because the chimera lacks the C-terminal subdomain of the auxilin clathrin-binding domain, it seemed possible that this subdomain is required for auxilin to act catalytically, and indeed its deletion caused auxilin to act stoichiometrically. In contrast, deletion of the N-terminal subdomain weakened auxilinclathrin binding and prevented auxilin from polymerizing clathrin. Therefore the C-terminal subdomain of the clathrin-binding domain of auxilin is required for auxilin to act catalytically, whereas the N-terminal subdomain strengthens auxilin-clathrin binding.Many of the functions carried out by the Hsc70 (the constitutive isoform of Hsp70) class of molecular chaperones both in vivo and in vitro require the presence of J-domain proteins (1, 2). For example, Hsc70 alone cannot uncoat clathrin-coated vesicles in vitro (3); the J-domain protein auxilin must also be present in catalytic amounts (4). Furthermore, knockout of auxilin in yeast or knockdown of auxilin in Caenorhabditis elegans prevents endocytosis from taking place (5-7), showing that aside from Hsc70, auxilin is also required for uncoating to take place in these organisms.J-domain proteins are thought to function by inducing substrates to bind to Hsc70, and this has been directly demonstrated for auxilin by showing that auxilin is required for ATP-dependent binding of Hsc70 at pH 6, where there is no uncoating (8). Although the mechanism of action of J-domain proteins is not completely understood, it is generally thought that they first bind substrate and then transfer the substrate to Hsc70-ATP and, finally, induce one round of rapid ATP hydrolysis by Hsc70 (9, 10). This stabilizes the binding of substrate because most substrates detach rapidly from Hsc70-ATP but very slowly from Hsc70-ADP (11)(12)(13)(14).To carry out their function J-domain proteins must contain both a J-domain that interacts with Hsc70 and a substratebinding domain that interacts with substrate. In fact, mammalian auxilin, a multi-domain protein, consists of at least three domains, an N-terminal Pten-like domain (15) of unknown function, a clathrin-binding domain, and...

Exchange of clathrin, AP2 and epsin on clathrin-coated pits in permeabilized tissue culture cells

“…Thus, Hsc70 and auxilin are slowly dissembling the clathrin lattice, rather than causing catastrophic collapse of the pits. These findings are consistent with our previous observations that, individual triskelions are able to be uncoated from clathrin baskets when a chimera of AP180 and auxilin is used (Ma et al, 2002).…”

A sequential mechanism for clathrin cage disassembly by 70-kDa heat-shock cognate protein (Hsc70) and auxilin

“…Using a simple perpendicular light-scattering assay we have measured the in vitro disassembly of clathrin cages by Hsc70 and auxilin, which occurs rapidly with a t 1∕2 of approximately 10 s. This is comparable to recent results from experiments that used dynamic light scattering to monitor clathrin cage disassembly (21,22) but faster than earlier centrifugation-based studies that had t 1∕2 values ranging from 2-10 min (19,20,24) but that also contained adaptor proteins such as AP180 or AP2, which are known to stabilize the cages and may consequently have slowed down disassembly. In this study, we have increased the time resolution beyond that of dynamic light scattering by monitoring perpendicular light scattering, and analysis of these data has allowed us to isolate and describe individual steps in the chaperone-mediated disassembly of cages that hitherto have remained invisible.…”

“…Previous binding studies have shown a maximal binding of three Hsc70s per triskelion at equilibrium (16,24), and, like in our study, this was shown to occur with only a single auxilin per triskelion. It has also been shown that approximately three molecules of Hsc70 dissociate one triskelion when coated vesicles rather than empty cages were used (6) and it has been found that, following disassembly, three Hsc70s are bound to each free triskelion (14,15).…”

Molecular and Functional Characterization of Clathrin- and AP-2-binding Determinants within a Disordered Domain of Auxilin

“…The clathrin-binding domain of auxilin (amino acids 547-813) is bipartite and consists of two subdomains comprising either predominantly clathrin N-terminal or distal domain interaction sites (15). Recently, Ma et al (16) constructed a chimeric protein from a C-terminal fragment of murine AP180 (residues 325-915) and the J-domain of auxilin. With this chimera, they were able to demonstrate that binding of a J-domain protein to the N-terminal domain of the clathrin heavy chain is sufficient to recruit hsc70 and to facilitate the dissociation of clathrin cages to a certain degree.…”