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

¹ Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, England, UK
² Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854
³ Cancer Research UK London Research Institute, London WC2A 3PX, England, UK

Correspondence to Elizabeth Smythe: e.smythe{at}sheffield.ac.uk

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 clathrin-coated 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 dominant-negative rab5^S34N, 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 efficiency. hRME-6 guanine nucleotide exchange factor activity requires hRME-6 binding to -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 rab5^S35N 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.

© 2008 Semerdjieva et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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