Functional analysis of AP-2 alpha and mu2 subunits.
The AP-2 adaptor complex plays a key role in cargo recognition and clathrin-coated vesicle formation at the plasma membrane. To investigate the functions of individual binding sites and domains of the AP-2 complex in vivo, we have stably transfected HeLa cells with wild-type and mutant small interfering RNA-resistant alpha and mu2 subunits and then used siRNA knockdowns to deplete the endogenous proteins. Mutating the PtdIns(4,5)P2 binding site of alpha, the phosphorylation site of mu2, or the YXXPhi binding site of mu2 impairs AP-2 function, as assayed by transferrin uptake. In contrast, removing the C-terminal appendage domain of alpha, or mutating the PtdIns(4,5)P2 binding site of mu2, has no apparent effect. However, adding a C-terminal GFP tag to alpha renders it completely nonfunctional. These findings demonstrate that there is some functional redundancy in the binding sites of the various AP-2 subunits, because no single mutation totally abolishes function. They also help to explain why GFP-tagged AP-2 never appears to leave the plasma membrane in some live cell imaging studies. Finally, they establish a new model system that can be used both for additional structure-function analyses, and as a way of testing tagged constructs for function in vivo.
Motley
Alison M
AM
Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 2XY, United Kingdom.
Berg
Nicola
N
Taylor
Marcus J
MJ
Sahlender
Daniela A
DA
Hirst
Jennifer
J
Owen
David J
DJ
Robinson
Margaret S
MS
D033965:Adaptor Protein Complex alpha Subunits / Q000737:chemistry / Q000378:metabolism*; D033983:Adaptor Protein Complex mu Subunits / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D004705:Endocytosis; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D014168:Transferrin / Q000378:metabolism
D033965:Adaptor Protein Complex alpha Subunits; D033983:Adaptor Protein Complex mu Subunits; D021122:Protein Subunits; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D014168:Transferrin; D049452:Green Fluorescent Proteins