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PMID: 18981233 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

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

The Journal of cell biology ·Vol. 183 ·No. 3 ·2008-11-03 ·Pages 499-511

Semerdjieva S, Shortt B, Maxwell E, Singh S, Fonarev P, Hansen J, Schiavo G, Grant BD, Smythe E

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 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 alpha-adaptin ear, which displaces the ear-associated mu2 kinase AAK1. siRNA-mediated depletion of hRME-6 increases phospho-mu2 levels, and expression of a phosphomimetic mu2 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)P(2)) 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 mu2 dephosphorylation and PtdIns(4,5)P(2) levels in CCVs.

MeSH Terms
Adaptor Protein Complex 2/physiology Animals Cell Line Cell Membrane/physiology Clathrin-Coated Vesicles/physiology Cytosol/physiology Endocytosis/physiology Humans Kidney Membrane Proteins/isolation & purification,physiology Mice Mice, Knockout Nerve Tissue Proteins/deficiency,physiology Neurons/physiology Phosphoric Monoester Hydrolases/deficiency,physiology rab5 GTP-Binding Proteins/physiology
Chemicals
Adaptor Protein Complex 2 Membrane Proteins Nerve Tissue Proteins synaptojanin Phosphoric Monoester Hydrolases rab5 GTP-Binding Proteins
Authors & Affiliations
9 authors, click to expand affiliations / ORCID
Semerdjieva Sophia
Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, England, UK.
Shortt Barry
Maxwell Emma
Singh Sukhdeep
Fonarev Paul
Hansen Jonathan
Schiavo Giampietro
Grant Barth D
Smythe Elizabeth
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
1540-8140
DOI
10.1083/jcb.200806016
Published
2008-11-03
Pages
499-511
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2575790
Subset
IM
Grants
British Heart Foundation · FS/2001053 · United Kingdom
Medical Research Council · G0300452 · United Kingdom
Biotechnology and Biological Sciences Research Council · BBS/B/04072 · United Kingdom
NIGMS NIH HHS · GM67237 · United States
NIGMS NIH HHS · R01 GM067237-06 · United States
NIGMS NIH HHS · R01 GM067237 · United States
Wellcome Trust · GR077544AIA · United Kingdom
External Links
PubMed source DOI: 10.1083/jcb.200806016 PMC Full Text View journal details
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