title: Coordinated regulation of AP2 uncoating from clathrin-coated vesicles by rab5 and hRME-6.
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.
authors.affiliation: Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, England, UK.
authors.forename: Sophia
authors.initials: S
authors.lastname: Semerdjieva
authors.forename: Barry
authors.initials: B
authors.lastname: Shortt
authors.forename: Emma
authors.initials: E
authors.lastname: Maxwell
authors.forename: Sukhdeep
authors.initials: S
authors.lastname: Singh
authors.forename: Paul
authors.initials: P
authors.lastname: Fonarev
authors.forename: Jonathan
authors.initials: J
authors.lastname: Hansen
authors.forename: Giampietro
authors.initials: G
authors.lastname: Schiavo
authors.forename: Barth D
authors.initials: BD
authors.lastname: Grant
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Smythe
mesh_terms: D033962:Adaptor Protein Complex 2 / Q000502:physiology*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D022163:Clathrin-Coated Vesicles / Q000502:physiology*; D003600:Cytosol / Q000502:physiology; D004705:Endocytosis / Q000502:physiology*; D006801:Humans; D007668:Kidney; D008565:Membrane Proteins / Q000302:isolation & purification / Q000502:physiology; D051379:Mice; D018345:Mice, Knockout; D009419:Nerve Tissue Proteins / Q000172:deficiency / Q000502:physiology; D009474:Neurons / Q000502:physiology*; D010744:Phosphoric Monoester Hydrolases / Q000172:deficiency / Q000502:physiology; D020696:rab5 GTP-Binding Proteins / Q000502:physiology*
chemical_list: D033962:Adaptor Protein Complex 2; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C097702:synaptojanin; D010744:Phosphoric Monoester Hydrolases; D020696:rab5 GTP-Binding Proteins