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MBC in Press, published online ahead of print October 31, 2007
Mol. Biol. Cell 10.1091/mbc.E07-09-0870

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Submitted on September 6, 2007
Revised on October 16, 2007
Accepted on October 19, 2007

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

Iris Rapoport, Werner Boll, Anan Yu, Till Böcking, and Tom Kirchhausen

Department of Cell Biology and Immune Disease Institute (IDI, formerly CBRI), Harvard Medical School, Boston, MA 02115

Monitoring Editor: Sandra Schmid

The 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 believed 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 a 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.

Address correspondence to: Tom Kirchhausen (kirchhausen{at}crystal.harvard.edu)






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