Clathrin coat disassembly by the yeast Hsc70/Ssa1p and auxilin/Swa2p proteins observed by single-particle burst analysis spectroscopy.

Abstract

The role of clathrin-coated vesicles in receptor-mediated endocytosis is conserved among eukaryotes, and many of the proteins required for clathrin coat assembly and disassembly have orthologs in yeast and mammals. In yeast, dozens of proteins have been identified as regulators of the multistep reaction required for endocytosis, including those that regulate disassembly of the clathrin coat. In mammalian systems, clathrin coat disassembly has been reconstituted using neuronal clathrin baskets mixed with the purified chaperone ATPase 70-kDa heat shock cognate (Hsc70), plus a clathrin-specific co-chaperone, such as the synaptic protein auxilin. Yet, despite previous characterization of the yeast Hsc70 ortholog, Ssa1p, and the auxilin-like ortholog, Swa2p, testing mechanistic models for disassembly of nonneuronal clathrin coats has been limited by the absence of a functional reconstitution assay. Here we use single-particle burst analysis spectroscopy, in combination with fluorescence correlation spectroscopy, to follow the population dynamics of fluorescently tagged yeast clathrin baskets in the presence of purified Ssa1p and Swa2p. An advantage of this combined approach for mechanistic studies is the ability to measure, as a function of time, changes in the number and size of objects from a starting population to the reaction products. Our results indicate that Ssa1p and Swa2p cooperatively disassemble yeast clathrin baskets into fragments larger than the individual triskelia, suggesting that disassembly of clathrin-coated vesicles may proceed through a partially uncoated intermediate.

• Authors: Kelly C Krantz, Jason Puchalla, Rajan Thapa, Callie Kobayashi, Margaret Bisher, Julie Viehweg, Chavela M Carr, Hays S Rye
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 288 Issue 37 Pg. 26721-30 (Sep 13 2013) ISSN: 1083-351X [Electronic] United States
• PMID: 23913685 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• Auxilins
• Clathrin
• HSP70 Heat-Shock Proteins
• Phosphoproteins
• SWA2 protein, S cerevisiae
• Saccharomyces cerevisiae Proteins
• Vesicular Transport Proteins
• Green Fluorescent Proteins
• Adenosine Triphosphate
• Adenosine Triphosphatases
• SSA1 protein, S cerevisiae

Topics

• Adenosine Triphosphatases (metabolism)
• Adenosine Triphosphate (chemistry)
• Auxilins (metabolism)
• Cell Membrane (metabolism)
• Chromatography, Gel
• Clathrin (metabolism)
• Cytoplasm (metabolism)
• Endocytosis
• Green Fluorescent Proteins (metabolism)
• HSP70 Heat-Shock Proteins (metabolism)
• Hydrolysis
• Microscopy, Electron
• Phosphoproteins (metabolism)
• Saccharomyces cerevisiae (metabolism)
• Saccharomyces cerevisiae Proteins (metabolism)
• Spectrophotometry
• Vesicular Transport Proteins (metabolism)