EpsinR is an adaptor for the SNARE protein Vti1b.
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 isolated CCVs from control and siRNA-treated cells and 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 approximately 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.
Hirst
Jennifer
J
University of Cambridge, Cambridge Institute for Medical Research, Cambridge CB2 2XY, United Kingdom.
Miller
Sharon E
SE
Taylor
Marcus J
MJ
von Mollard
Gabriele Fischer
GF
Robinson
Margaret S
MS
D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D046529:Microscopy, Electron, Transmission; D011485:Protein Binding; D050766:Qb-SNARE Proteins; D034741:RNA, Small Interfering; D050600:SNARE Proteins; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000378:metabolism*
D033942:Adaptor Proteins, Vesicular Transport; C475910:CLINT1 protein, human; D002352:Carrier Proteins; D008565:Membrane Proteins; D050766:Qb-SNARE Proteins; D034741:RNA, Small Interfering; D050600:SNARE Proteins; D018808:Transcription Factor AP-1; C491419:VTI1A protein, human; C491416:VTI1B protein, human; D033921:Vesicular Transport Proteins