Abstract
Uncoating of clathrin-coated vesicles requires the J-domain protein auxilin for targeting hsc70 to the clathrin coats and for stimulating the hsc70 ATPase activity. This results in the release of hsc70-complexed clathrin triskelia and concomitant dissociation of the coat. To understand the complex role of auxilin in uncoating and clathrin assembly in more detail, we analyzed the molecular organization of its clathrin-binding domain (amino acids 547-813). CD spectroscopy of auxilin fragments revealed that the clathrin-binding domain is almost completely disordered in solution. By systematic mapping using synthetic peptides and by site-directed mutagenesis, we identified short peptide sequences involved in clathrin heavy chain and AP-2 binding and evaluated their significance for the function of auxilin. Some of the binding determinants, including those containing sequences 674DPF and 636WDW, showed dual specificity for both clathrin and AP-2. In contrast, the two DLL motifs within the clathrin-binding domain were exclusively involved in clathrin binding. Surprisingly, they interacted not only with the N-terminal domain of the heavy chain, but also with the distal domain. Moreover, both DLL peptides proved to be essential for clathrin assembly and uncoating. In addition, we found that the motif 726NWQ is required for efficient clathrin assembly activity. Auxilin shares a number of protein-protein interaction motifs with other endocytic proteins, including AP180. We demonstrate that AP180 and auxilin compete for binding to the alpha-ear domain of AP-2. Like AP180, auxilin also directly interacts with the ear domain of beta-adaptin. On the basis of our data, we propose a refined model for the uncoating mechanism of clathrin-coated vesicles.
Highlights
According to the current model for the uncoating of clathrincoated vesicles, it is believed that auxilin first binds to clathrin coats and recruits hsc[70] in an ATP-dependent reaction via its J-domain
Because auxilin is able to stimulate the hsc70-dependent dissociation of such truncated clathrin cages, it has been suggested that the interaction of auxilin with the proximal or distal domain of clathrin is sufficient for its cofactor activity (1)
Computer-based primary structure analysis predicts very little secondary structure for the entire clathrin-binding domain of auxilin. This lack of organized structure seems reminiscent of the disordered clathrinbinding domains of epsin-1 and AP180, two accessory proteins involved in the formation of clathrin-coated vesicles (13, 18)
Summary
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 hsc[70] and to facilitate the dissociation of clathrin cages to a certain degree. To characterize the overall structural organization of the clathrin-binding domain, we employed CD spectroscopy, which showed that this segment of auxilin is intrinsically disordered. This finding formed the basis for further dissections of the clathrin-binding domain and for defined mutations within putative protein-protein interaction motifs. The 726NWQ sequence was recognized as important for efficient clathrin assembly by auxilin
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