title: 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.affiliation: From the Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544.
authors.forename: Kelly C
authors.initials: KC
authors.lastname: Krantz
authors.forename: Jason
authors.initials: J
authors.lastname: Puchalla
authors.forename: Rajan
authors.initials: R
authors.lastname: Thapa
authors.forename: Callie
authors.initials: C
authors.lastname: Kobayashi
authors.forename: Margaret
authors.initials: M
authors.lastname: Bisher
authors.forename: Julie
authors.initials: J
authors.lastname: Viehweg
authors.forename: Chavela M
authors.initials: CM
authors.lastname: Carr
authors.forename: Hays S
authors.initials: HS
authors.lastname: Rye
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000737:chemistry; D034021:Auxilins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D002966:Clathrin / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004705:Endocytosis; D049452:Green Fluorescent Proteins / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006868:Hydrolysis; D008854:Microscopy, Electron; D010750:Phosphoproteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013053:Spectrophotometry; D033921:Vesicular Transport Proteins / Q000378:metabolism*
chemical_list: D034021:Auxilins; D002966:Clathrin; D018840:HSP70 Heat-Shock Proteins; D010750:Phosphoproteins; C419865:SWA2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C095973:SSA1 protein, S cerevisiae
keywords: Auxilin; Burst Analysis Spectroscopy; Clathrin; Endocytosis; Fluorescence; Hsc70; Membrane Trafficking; Molecular Chaperone; Single-particle Analysis
references.citation: Curr Biol. 2000 Nov 2;10(21):1349-58
references.pmid: 11084334
references.citation: Nat Cell Biol. 2011 Dec 22;14(1):2-10
references.pmid: 22193158
references.citation: Mol Cell. 2008 Jul 25;31(2):232-43
references.pmid: 18550409
references.citation: Proc Natl Acad Sci U S A. 2008 Sep 23;105(38):14400-5
references.pmid: 18780782
references.citation: Nature. 2004 Dec 2;432(7017):649-53
references.pmid: 15502813
references.citation: J Biol Chem. 1991 Oct 15;266(29):19565-71
references.pmid: 1833403
references.citation: J Biol Chem. 2000 Jan 14;275(2):1365-70
references.pmid: 10625686
references.citation: Mol Biol Cell. 2008 Jan;19(1):405-13
references.pmid: 17978091
references.citation: Annu Rev Cell Dev Biol. 2010;26:137-56
references.pmid: 19575650
references.citation: Proc Natl Acad Sci U S A. 2006 Jul 5;103(27):10265-10270
references.pmid: 16798879
references.citation: Mol Biol Cell. 2008 Jul;19(7):2830-43
references.pmid: 18434597
references.citation: Dev Cell. 2009 Sep;17(3):403-16
references.pmid: 19758564
references.citation: Arch Biochem Biophys. 2008 Oct 15;478(2):167-74
references.pmid: 18706386
references.citation: PLoS Biol. 2011 Mar;9(3):e1001037
references.pmid: 21445329
references.citation: Nature. 1997 Aug 21;388(6644):792-8
references.pmid: 9285593
references.citation: Nat Rev Mol Cell Biol. 2013 Jun;14(6):382-92
references.pmid: 23698585
references.citation: Nat Cell Biol. 2001 Feb;3(2):215-9
references.pmid: 11175756
references.citation: Nat Struct Mol Biol. 2011 Mar;18(3):295-301
references.pmid: 21278753
references.citation: Nat Cell Biol. 2000 Dec;2(12):958-63
references.pmid: 11146663
references.citation: Nature. 2011 May 12;473(7346):181-6
references.pmid: 21532587
references.citation: Nature. 2003 Oct 16;425(6959):686-91
references.pmid: 14562095
references.citation: Mol Cell. 2005 Nov 23;20(4):513-24
references.pmid: 16307916
references.citation: Trends Cell Biol. 2012 Jan;22(1):1-13
references.pmid: 22018597
references.citation: J Biol Chem. 2000 Mar 24;275(12):8439-47
references.pmid: 10722678
references.citation: Nature. 1995 Dec 7;378(6557):632-5
references.pmid: 8524399
references.citation: Nat Rev Mol Cell Biol. 2011 Jul 22;12(8):517-33
references.pmid: 21779028
references.citation: J Cell Biol. 1984 Aug;99(2):734-41
references.pmid: 6146631
references.citation: EMBO J. 2012 Nov 5;31(21):4221-35
references.pmid: 22990239
references.citation: J Biol Chem. 2008 Nov 14;283(46):32003-13
references.pmid: 18782766
references.citation: Proc Natl Acad Sci U S A. 2011 Apr 26;108(17):6927-32
references.pmid: 21482805
references.citation: Eur J Cell Biol. 2000 May;79(5):336-42
references.pmid: 10887964
references.citation: J Biol Chem. 1990 Apr 25;265(12):6682-7
references.pmid: 1969864
references.citation: Cell. 1983 Mar;32(3):921-9
references.pmid: 6131746
references.citation: Traffic. 2011 Dec;12(12):1868-78
references.pmid: 21883765
references.citation: Nature. 2004 Dec 2;432(7017):573-9
references.pmid: 15502812
references.citation: Biochemistry. 2006 May 9;45(18):5916-22
references.pmid: 16669634
references.citation: J Mol Biol. 2004 Feb 13;336(2):461-71
references.pmid: 14757058
references.citation: Traffic. 2007 Sep;8(9):1137-44
references.pmid: 17645434
references.citation: Mol Biol Cell. 2001 Dec;12(12):3773-82
references.pmid: 11739779
references.citation: J Cell Biol. 1990 Jul;111(1):19-29
references.pmid: 1973169
references.citation: J Biol Chem. 1994 Mar 4;269(9):6931-9
references.pmid: 8120055
references.citation: J Biol Chem. 1985 Aug 25;260(18):10044-9
references.pmid: 2862146
references.citation: J Cell Biol. 1984 Aug;99(2):723-33
references.pmid: 6146630
references.citation: Mol Biol Cell. 2009 Nov;20(21):4563-74
references.pmid: 19741093
references.citation: J Biol Chem. 2002 Dec 20;277(51):49267-74
references.pmid: 12377777
references.citation: EMBO J. 2010 Feb 3;29(3):655-65
references.pmid: 20033059
references.citation: EMBO J. 1992 Aug;11(8):2811-8
references.pmid: 1639056