Visualization of the binding of Hsc70 ATPase to clathrin baskets: implications for an uncoating mechanism.
Clathrin assembly into coated pits and vesicles is promoted by accessory proteins such as auxilin and AP180, and disassembly is effected by the Hsc70 ATPase. These interactions may be mimicked in vitro by the assembly and disassembly of clathrin "baskets." The chimera C58J is a minimal construct capable of supporting both reactions; it consists of the C58 moiety of AP180, which facilitates clathrin assembly, fused with the J domain of auxilin, which recruits Hsc70 to baskets. We studied the process of disassembly by using cryo-electron microscopy to identify the initial binding site of Hsc70 on clathrin-C58J baskets at pH 6, under which conditions disassembly does not proceed further. Hsc70 interactions involve two sites: (i) its major interaction is with the sides of spars of the clathrin lattice, close to the triskelion hubs and (ii) there is another interaction at a site at the N-terminal hooks of the clathrin heavy chains, presumably via the J domain of C58J. We propose that individual triskelions may be extricated from the clathrin lattice by the concerted action of up to six Hsc70 molecules, which intercalate between clathrin leg segments, prying them apart. Three Hsc70s remain bound to the dissociated triskelion, close to its trimerization hub.
Heymann
J Bernard
JB
Laboratory of Structural Biology, NIAMS, National Institutes of Health, Bethesda, MD, USA.
Iwasaki
Kenji
K
Yim
Yang-In
YI
Cheng
Naiqian
N
Belnap
David M
DM
Greene
Lois E
LE
Eisenberg
Evan
E
Steven
Alasdair C
AC
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D034021:Auxilins; D001665:Binding Sites; D002417:Cattle; D002966:Clathrin / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000737:chemistry* / Q000378:metabolism; D020285:Cryoelectron Microscopy; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D034041:Monomeric Clathrin Assembly Proteins; D046912:Multiprotein Complexes / Q000737:chemistry; D011993:Recombinant Fusion Proteins
D034021:Auxilins; D002966:Clathrin; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D034041:Monomeric Clathrin Assembly Proteins; D046912:Multiprotein Complexes; D011993:Recombinant Fusion Proteins; C439023:clathrin assembly protein AP180; D000251:Adenosine Triphosphatases