A Motif in the Clathrin Heavy Chain Required for the Hsc70/Auxilin Uncoating Reaction

Abstract

The 70-kDa heat-shock cognate protein (Hsc70) chaperone is an ATP-dependent "disassembly enzyme" for many subcellular structures, including clathrin-coated vesicles where it functions as an uncoating ATPase. Hsc70, and its cochaperone auxilin together catalyze coat disassembly. Like other members of the Hsp70 chaperone family, it is thought that ATP-bound Hsc70 recognizes the clathrin triskelion through an unfolded exposed hydrophobic segment. The best candidate is the unstructured C terminus (residues 1631-1675) of the heavy chain at the foot of the tripod below the hub, containing the sequence motif QLMLT, closely related to the sequence bound preferentially by the substrate groove of Hsc70 (Fotin et al., 2004b). To test this hypothesis, we generated in insect cells recombinant mammalian triskelions that in vitro form clathrin cages and clathrin/AP-2 coats exactly like those assembled from native clathrin. We show that coats assembled from recombinant clathrin are good substrates for ATP-and auxilin-dependent, Hsc70-catalyzed uncoating. Finally, we show that this uncoating reaction proceeds normally when the coats contain recombinant heavy chains truncated C-terminal to the QLMLT motif, but very inefficiently when the motif is absent. Thus, the QLMLT motif is required for Hsc-70 -facilitated uncoating, consistent with the proposal that this sequence is a specific target of the chaperone. INTRODUCTIONClathrin-coated vesicles, the best known carrier of intracellular membrane traffic, transport proteins and lipids from the plasma membrane to endosomes and between endosomes and the trans-Golgi network. Polymerization of the principal coat protein clathrin into a lattice-like assembly by sequential addition of individual, cytosolic clathrin trimers to a growing shell shapes the budding coated pit (Ehrlich et al., 2004; for review, see Kirchhausen, 2000). Intermediary proteins, known as adaptors, form the interface between the outer clathrin coat and the incorporated membrane bilayer. These adaptors selectively recruit membrane-anchored proteins ("cargo"). The most prominent adaptors are the heterotetrameric AP-2 and its cousins AP-1, AP-3, and AP-4, but there are other, presumably more specialized, adaptors, such as ␤-arrestins, epsin, GGAs, and dishevelled (Yu et al., 2007; for review, see Owen et al., 2004;Robinson, 2004).Coats can assemble in vitro without incorporated membrane, either from clathrin alone ("cages", stable only at reduced pH) or from clathrin plus heteroteterameric APs ("coats", stable at neutral pH) (Keen et al., 1979;Kirchhausen and Harrison, 1981; Vigers et al., 1986a,b;Kirchhausen, 2000;Fotin et al., 2004b). Coat assembly in vitro can proceed to completion without intervention of other factors. In vivo, coat assembly occurs only on membrane surfaces, and fission (pinching off) of the incorporating bilayer requires the large GTPase, dynamin (Sever, 2002;Praefcke and McMahon, 2004;Kruchten and McNiven, 2006;Macia et al., 2006). Removal of the coat (uncoating), essential for subsequent vesicle f...

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

“…1b), near known binding sites of auxilin and Hsc70 (20). Furthermore, a C-terminal clathrin heavy chain mutation, or truncation, resulted in defective coat disassembly (18,20). Two observations suggest that HRY54 CHC1-GFP forms functional CCVs, in vivo.…”

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

“…It has also been shown that approximately three molecules of Hsc70 dissociate one triskelion when coated vesicles rather than empty cages were used (6) and it has been found that, following disassembly, three Hsc70s are bound to each free triskelion (14,15). Identification of the Hsc70 binding motif and the location of this within the cage structure suggest that Hsc70 can bind to three potential binding sites on flexible regions protruding down from the hub (27).…”

Molecular Structure, Function, and Dynamics of Clathrin-Mediated Membrane Traffic

“…1) (Fotin et al 2004b). The carboxy-terminal segment contains a short region with an amino acid sequence that is preferentially recognized by Hsc70 and required for Hsc70-facilitated uncoating (Rapoport et al 2008;Böcking et al 2011). The 42 zig-zags that make up the bulk of the polypeptide chain create an extended, gently curved "leg" for the clathrin triskelion (from the Greek for "a three-legged structure"), with the b-propeller "terminal domain" at its tip.…”

“…Within the clathrin carboxy-terminal segment is a short, hydrophobic sequence, necessary for Hsc70-and ATP-dependent uncoating and very similar to an optimal Hsc70-interacting peptide (Rapoport et al 2008). Auxilin associates with a clathrin lattice in such a way that an Hsc70 molecule, recruited by the J-domain, can, in turn, bind a nearby clathrin carboxyterminal segment (Fig.…”