A motif in the clathrin heavy chain required for the Hsc70/auxilin uncoating reaction.
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.
Rapoport
Iris
I
Department of Cell Biology and Immune Disease Institute, Harvard Medical School, Boston, MA 02115, USA.
Boll
Werner
W
Yu
Anan
A
Böcking
Till
T
Kirchhausen
Tom
T
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D034021:Auxilins / Q000378:metabolism*; D002417:Cattle; D033922:Clathrin Heavy Chains / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D050883:HSC70 Heat-Shock Proteins / Q000378:metabolism*; D007313:Insecta; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship
D034021:Auxilins; D050883:HSC70 Heat-Shock Proteins; D010455:Peptides; D011994:Recombinant Proteins; D033922:Clathrin Heavy Chains