The Library

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

Tools

Rothnie, Alice, Clarke, Anthony R., Kuzmic, Petr, Cameron, Angus and Smith, Corinne. (2011) A sequential mechanism for clathrin cage disassembly by 70-kDa heat-shock cognate protein (Hsc70) and auxilin. National Academy of Sciences. Proceedings, Vol.108 (No.17). pp. 6927-6932. ISSN 0027-8424

Preview

PDF
WRAP_Smith_-rothnie_et_el_280211.pdf - Requires a PDF viewer such as GSview, Xpdf or Adobe Acrobat Reader
Download (6Mb)

Official URL: http://dx.doi.org/10.1073/pnas.1018845108

Abstract

An essential stage in endocytic coated vesicle recycling is the dissociation of clathrin from the vesicle coat by the molecular chaperone, Hsc70 and the J-domain-containing protein, auxilin, in an ATP-dependent process. We present the first detailed mechanistic analysis of clathrin disassembly catalysed by Hsc70 and auxilin, using loss of perpendicular light scattering to monitor the process. We report that a single auxilin per clathrin triskelion is required for maximal rate of disassembly, that ATP is hydrolysed at the same rate that disassembly occurs and that three ATP molecules are hydrolysed per clathrin triskelion released. Stopped-flow measurements revealed a lag phase in which the scattering intensity increased owing to association of Hsc70 with clathrin cages followed by serial rounds of ATP hydrolysis prior to triskelion removal. Global fit of stopped flow data to several physically plausible mechanisms showed the best fit to a model in which sequential hydrolysis of three separate ATP molecules is required for the eventual release of a triskelion from the clathrin-auxilin cage.

Item Type:	Journal Article
Subjects:	Q Science > QH Natural history
Divisions:	Faculty of Science > Life Sciences (2010- ) > Biological Sciences ( -2010) Faculty of Science > Life Sciences (2010- )
Library of Congress Subject Headings (LCSH):	Coated vesicles, Endocytosis, Adenosine triphosphate
Journal or Publication Title:	National Academy of Sciences. Proceedings
Publisher:	National Academy of Sciences
ISSN:	0027-8424
Date:	26 April 2011
Volume:	Vol.108
Number:	No.17
Page Range:	pp. 6927-6932
Identification Number:	10.1073/pnas.1018845108
Status:	Peer Reviewed
Publication Status:	Published
Access rights to Published version:	Restricted or Subscription Access
Funder:	Medical Research Council (Great Britain) (MRC), Wellcome Trust (London, England)
Grant number:	G0601125 (MRC), 055663/Z/98/Z (WT)
References:	1. Grant BD & Donaldson JG (2009) Pathways and mechanisms of endocytic recycling Nature Rev. Mol. Cell. Biol. 10, 597-608. 2. Brodsky FM, Chen CY, Knuehl C, Towler MC, & Wakeham DE (2001) Biological basket weaving: formation and function of clathrin-coated vesicles Annu. Rev. Cell. Dev. Biol. 17, 517-556. 3. Kirchhausen T (2000) Clathrin Annu. Rev. Biochem. 69, 699-727. 4. Schmid SL (1997) Clathrin-coated vesicle formation and protein sorting: an integrated process Annu. Rev. Biochem. 66, 511-548. 5. Pearse BM & Robinson MS (1990) Clathrin, adaptors, and sorting Annu. Rev. Cell Biol. 6, 151-171. 6. Eisenberg E & Greene LE (2007) Multiple roles of auxilin and hsc70 in clathrinmediated endocytosis Traffic 8, 640-646. 7. Holstein SE, Ungewickell H, & Ungewickell E (1996) Mechanism of clathrin basket dissociation: separate functions of protein domains of the DnaJ homologue auxilin J. Cell. Biol. 135, 925-937. 8. Ungewickell E, Ungewickell H, & Holstein SE (1997) Functional interaction of the auxilin J domain with the nucleotide- and substrate-binding modules of Hsc70 J. Biol. Chem. 272, 19594-19600. 9. Barouch W, Prasad K, Greene LE, & Eisenberg E (1997) Auxilin-induced interaction of the molecular chaperone Hsc70 with clathrin baskets Biochemistry 36, 4303-4308. 10. Jiang RF, Greener T, Barouch W, Greene L, & Eisenberg E (1997) Interaction of auxilin with the molecular chaperone, Hsc70 J. Biol. Chem. 272, 6141-6145. 11. Gao B, Greene L, & Eisenberg E (1994) Characterization of nucleotide-free uncoating ATPase and its binding to ATP, ADP, and ATP analogues Biochemistry 33, 2048-2054. 12. Ha JH & McKay DB (1994) ATPase kinetics of recombinant bovine 70 kDa heat shock cognate protein and its amino-terminal ATPase domain Biochemistry 33, 14625- 14635. 13. Greene LE & Eisenberg E (1990) Dissociation of clathrin from coated vesicles by the uncoating ATPase J. Biol. Chem. 265, 6682-6687. 14. Schlossman DM, Schmid SL, Braell WA, & Rothman JE (1984) An enzyme that removes clathrin coats: purification of an uncoating ATPase J. Cell. Biol. 99, 723-733. 15. Prasad K, Heuser J, Eisenberg E, & Greene L (1994) Complex formation between clathrin and uncoating ATPase J. Biol. Chem. 269, 6931-6939. 16. Ungewickell E, Ungewickell H, Holstein SEH, Lindner R, Prasad K, et al. (1995) Role of auxilin in uncoating clathrin-coated vesicles Nature 378, 632-635. 17. Xing Y, Böcking T, Wolf M, Grigorieff N, Kirchhausen T et al. (2010) Structure of clathrin coat with bound Hsc70 and auxilin: mechanism of Hsc70-facilitated disassembly EMBO J. 29, 655-665. 18. Fotin A, Cheng Y, Grigorieff N, Walz T, Harrison SC, et al. (2004) Structure of an auxilin-bound clathrin coat and its implications for the mechanism of uncoating Nature 432, 649-653. 19. Barouch W, Prasad K, Greene LE, & Eisenberg E (1994) ATPase activity associated with the uncoating of clathrin baskets by Hsp70 J. Biol. Chem. 269, 28563-28568. 20. Prasad K, Barouch W, Greene L, & Eisenberg E (1993) A protein cofactor is required for uncoating of clathrin baskets by uncoating ATPase J. Biol. Chem. 268, 23758- 23761. 21. Jiang J, Prasad K, Lafer EM, & Sousa R (2005) Structural basis of interdomain communication in the Hsc70 chaperone Moll. Cell. 20, 513-524. 22. Schuermann JP, Jiang J, Cuellar J, Llorca O, Wang L, et al. (2008) Structure of the Hsp110:Hsc70 nucleotide exchange machine Moll. Cell. 31, 232-243. 23. Myung JI & Pitt MA (2004) Model comparison methods Meth. Enzymol. 383, 351-366. 24. Ma Y, Greener T, Pacold ME, Kaushal S, Greene L, et al. (2002) Identification of domain required for catalytic activity of auxilin in supporting clathrin uncoating by Hsc70 J. Biol. Chem. 277, 49267-49274. 25. Jiang R, Gao B, Prasad K, Greene LE, & Eisenberg E (2000) Hsc70 chaperones clathrin and primes it to interact with vesicle membranes J Biol Chem 275, 8439-8447. 26. Ahle S & Ungewickell E (1990) Auxilin, a newly identified clathrin-associated protein in coated vesicles from bovine brain J. Cell. Biol. 111, 19-29. 27. Rapoport I, Boll W, Yu A, Bocking T, & Kirchhausen T (2008) A motif in the clathrin heavy chain required for the Hsc70/auxilin uncoating reaction Mol. Biol. Cell. 28. Mayer MP & Bukau B (2005) Hsp70 chaperones: cellular functions and molecular mechanism Cell. Mol. Life. Sci. 62, 670-684. 29. Kuzmic P (1996) Program DYNAFIT for the analysis of enzyme kinetic data: Application to HIV proteinase Anal. Biochem. 237, 260-273. 30. Kuzmic P (2009) DynaFit - A software package for enzymology Meth. Enzymol. 467, 247-280. 31. Burnham KB & Anderson DR (2002) Model Selection and Multimodel Inference: A Practical Information-Theoretic Approach (Springer-Verlag, New York). 32. Bates DM & Watts DG (1988) Nonlinear Regression Analysis and its Applications (Wiley, New York). 33. Brooks I, Watts DG, Soneson KK, & Hensley P (1994) Determining confidence intervals for parameters derived from analysis of equilibrium analytical ultracentrifugation data Meth. Enzymol. 240, 459-478.
URI:	http://wrap.warwick.ac.uk/id/eprint/4548