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Published online 5 February 2001. doi:10.1083/jcb.152.3.607
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© The Rockefeller University Press, 0021-9525/2001//607 $5.00
The Journal of Cell Biology, Volume 152, Number 3, , 2001 607-620

Original Article

Dominant-Interfering Hsc70 Mutants Disrupt Multiple Stages of the Clathrin-Coated Vesicle Cycle in Vivo



Sherri L. Newmyera and Sandra L. Schmida

a Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037
Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037.(858) 784-9126(858) 784-2311

slschmid{at}scripps.edu

Within the clathrin-coated vesicle (CCV) cycle, coat assembly drives the internalization of receptors from the cell surface and disassembly allows for the processing of internalized ligands. The heat shock cognate protein, hsc70, has been implicated in regulating coat disassembly. We find that in cells overexpressing ATPase-deficient hsc70 mutants, uncoating of CCVs is inhibited in vivo, and the majority of unassembled cytosolic clathrin shifts to an assembled pool that cofractionates with AP1 and AP2. Surprisingly, this assembled pool of coat proteins accumulates in the absence of cargo receptors, suggesting that disruption of hsc70 activity may cause misassembly of empty clathrin cages. The strongest effect of overexpression of hsc70 mutants is a block in transferrin receptor (TfnR) recycling, which cannot be accounted for by the degree of inhibition of uncoating of endocytic CCVs. These results suggest that hsc70 participates in multiple transport and/or sorting events between endosomal compartments. Additionally, the mutant-expressing cells are defective at internalizing transferrin. In the most potent case, the initial rate of uptake is inhibited 10-fold, and TfnR levels double at the cell surface. Our findings demonstrate that hsc70 indeed regulates coat disassembly and also suggest that this chaperone broadly modulates clathrin dynamics throughout the CCV cycle.

Key Words: Hsc70 • endocytosis • clathrin-coated vesicles • transferrin receptor • recycling

© 2001 The Rockefeller University Press

The online version of this article contains supplemental material.

Abbreviations used in this paper: AP, adaptor protein; BXX-Tfn and BSS-Tfn, biotinylated Tfn; CCV, clathrin-coated vesicle; Endo H, endoglycosidase H; HA, hemagglutinin; MPR, 300-kD mannose 6-phosphate receptor; PM, plasma membrane; RME, receptor-mediated endocytosis; Tfn, transferrin; TfnR, Tfn receptor.


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