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

Author:

Semerdjieva Sophia1,Shortt Barry1,Maxwell Emma1,Singh Sukhdeep1,Fonarev Paul2,Hansen Jonathan1,Schiavo Giampietro3,Grant Barth D.2,Smythe Elizabeth1

Affiliation:

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

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 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 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 efficiency. 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.

Publisher

Rockefeller University Press

Subject

Cell Biology

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