A yeast DNA J protein required for uncoating of clathrin-coated vesicles in vivo

Abstract

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 Coat Disassembly by the Yeast Hsc70/Ssa1p and Auxilin/Swa2p Proteins Observed by Single-particle Burst Analysis Spectroscopy

“…Furthermore, a recent mechanistic study of individual baskets has revealed a cooperative uncoating mechanism under single turnover conditions, with Hsc70 and auxilin prebound to baskets tethered to a coverslip (20). Although genetic evidence from S. cerevisiae has confirmed that the Hsc70 ortholog, Ssa1p, and the auxilin ortholog, Swa2p, are necessary for clathrin uncoating (4,5,22), it has not been determined whether these proteins are sufficient for yeast clathrin coat disassembly in a reconstituted system. To examine the mechanism of clathrin uncoating in yeast, we analyzed the single-particle dynamics of clathrin coat disassembly, in free solution, reconstituted from purified components.…”

“…Biochemical research using purified neuronal proteins has demonstrated that clathrin coats (hereafter referred to as "baskets," when membrane is absent), Hsc70, auxilin, and ATP comprise the minimal system to reconstitute clathrin uncoating in vitro (4,8,10,14). Furthermore, a recent mechanistic study of individual baskets has revealed a cooperative uncoating mechanism under single turnover conditions, with Hsc70 and auxilin prebound to baskets tethered to a coverslip (20).…”

“…Clathrin baskets were obtained by cell fractionation, using differential centrifugation and size exclusion chromatography (4,5). Small vesicles and GFP-tagged clathrin baskets, enriched in the pellet of a 100,000 ϫ g centrifugation step, were further fractionated on a S1000 gel filtration column, resulting in a large, Chc1p-GFP-positive peak (Fig.…”

“…More recent work using advanced imaging methods has suggested, by contrast, that clathrin coat disassembly occurs through a cooperative mechanism (20). Whereas purified yeast orthologs of Hsc70 and auxilin display disassembly activity toward neuronal clathrin coats in vitro (4,22), to date they have displayed no disassembly activity toward yeast-derived clathrin coats, based on the absence of triskelia in the supernatant fraction of centrifugation assays. Our results indicate that the core Hsc70-auxilin machinery in yeast, Ssa1p-Swa2p, is, in fact, sufficient to initiate clathrin coat removal in yeast cells.…”

“…One possible hurdle is purification of fully active and/or complete disassembly machinery. The Hsc70, Ssa1p, with the auxilin-like cofactor, Swa2p, were shown to have ATPase activity and to bind clathrin (4,5); however, stimulation of yeast clathrin disassembly was not detectable using a centrifugation-based assay. It is possible that, unlike in neurons, the core clathrin disassembly machinery in yeast requires additional factors.…”