Coordinated regulation of AP2 uncoating from clathrin-coated vesicles by rab5 and hRME-6

Sophia Semerdjieva, Barry Shortt, Emma Maxwell, Sukhdeep Singh, Paul Fonarev, Jonathan Hansen, Giampietro Schiavo, Barth D. Grant, Elizabeth Smythe

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73 Scopus citations

Abstract

Here we investigate the role of rab5 and its cognate exchange factors rabex-5 and hRME-6 in the regulation of AP2 uncoating from endocytic clathrincoated vesicles (CCVs). In vitro , we show that the rate of AP2 uncoating from CCVs is dependent on the level of functional rab5. In vivo, overexpression of dominantnegative rab5S34N , or small interfering RNA (siRNA) - mediated depletion of hRME-6, but not rabex-5, resulted in increased steady-state levels of AP2 associated with endocytic vesicles, which is consistent with reduced uncoating effi ciency. hRME-6 guanine nucleotide exchange factor activity requires hRME-6 binding to α-adaptin ear, which displaces the ear-associated μ2 kinase AAK1. siRNA-mediated depletion of hRME-6 increases phospho-μ2 levels, and expression of a phosphomimetic μ2 mutant increases levels of endocytic vesicle-associated AP2. Depletion of hRME-6 or rab5S35N expression also increases the levels of phosphoinositide 4,5-bisphosphate (PtdIns(4,5)P2 ) associated with endocytic vesicles. These data are consistent with a model in which hRME-6 and rab5 regulate AP2 uncoating in vivo by coordinately regulating μ2 dephosphorylation and PtdIns(4,5)P2 levels in CCVs.

Original languageEnglish (US)
Pages (from-to)499-511
Number of pages13
JournalJournal of Cell Biology
Volume183
Issue number3
DOIs
StatePublished - Nov 3 2008
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Cell Biology

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