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MBC in Press, published online ahead of print September 29, 2004
Mol. Biol. Cell 10.1091/mbc.E04-06-0468

A more recent version of this article appeared on December 1, 2004 Originally published as MBC in Press, 10.1091/mbc.E04-06-0468 on September 15, 2004
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Submitted on June 10, 2004
Accepted on September 2, 2004

EpsinR Is an Adaptor for the SNARE Protein Vti1b

Jennifer Hirst,* Sharon E. Miller,* Marcus J. Taylor,* Gabriele Fischer von Mollard,{dagger} and Margaret S. Robinson*{ddagger}

*University of Cambridge, CIMR, Cambridge CB2 2XY, United Kingdom; {dagger}Georg-August-Universität, Göttingen, Germany

Monitoring Editor: Suzanne Pfeffer

EpsinR is a clathrin-coated vesicle (CCV)-associated protein that binds to vti1b, suggesting that it may be a vti1b-selective adaptor. Depletion of epsinR to undetectable levels in HeLa cells using siRNA causes vti1b to redistribute from the perinuclear region to the cell periphery, but vti1a also redistributes in epsinR-depleted cells, and both vti isoforms redistribute in AP-1-depleted cells. As a more direct assay for epsinR function, we have isolated CCVs from control and siRNA-treated cells, then looked for differences in cargo content. In clathrin-depleted cells, both coat and cargo proteins are greatly reduced in this preparation. Knocking down epsinR causes a ~50% reduction in the amount of AP-1 copurifying with CCVs and vice versa, indicating that the two proteins are dependent on each other for maximum incorporation into the coat. In addition, vti1b, but not vti1a, is reduced by >70% in CCVs from both epsinR- and AP-1 depleted cells. Because AP-1 knockdown reduces the amount of epsinR in CCVs, it is possible that its effect on vti1b may be indirect. These findings provide in vivo evidence that epsinR is an adaptor for vti1b, and they also show that CCV isolation can be used as an assay for adaptor function.

{ddagger}Corresponding author. E-mail: msr12{at}mole.bio.cam.ac.uk






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