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Vol. 17, Issue 12, 5298-5308, December 2006

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Functional Analysis of AP-2 and µ2 Subunits

Alison M. Motley^*, Nicola Berg, Marcus J. Taylor, Daniela A. Sahlender, Jennifer Hirst, David J. Owen, and Margaret S. Robinson

Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 2XY, United Kingdom

Submitted May 24, 2006; Revised August 30, 2006; Accepted October 3, 2006
Monitoring Editor: Sandra Schmid

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 and µ2 subunits and then used siRNA knockdowns to deplete the endogenous proteins. Mutating the PtdIns(4,5)P2 binding site of , the phosphorylation site of µ2, or the YXX binding site of µ2 impairs AP-2 function, as assayed by transferrin uptake. In contrast, removing the C-terminal appendage domain of , or mutating the PtdIns(4,5)P2 binding site of µ2, has no apparent effect. However, adding a C-terminal GFP tag to 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.

Present addresses: * Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom;

Cambridge Centre for Brain Repair, Cambridge CB2 2PY, United Kingdom;

MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom.

Address correspondence to: Margaret S. Robinson (msr12{at}mole.bio.cam.ac.uk)

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