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A more recent version of this article appeared on December 1, 2008
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Submitted on July 11, 2008
Revised on September 19, 2008
Accepted on September 30, 2008
2 Appendage Scaffolds Alternate Cargo Endocytosis
*Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261; Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110; ||Children’s Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213; ¶Department of Pediatric Dentistry and The Carolina Center for Genome Science, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
Monitoring Editor: Sandra Lemmon
The independently-folded appendages of the large 
and 
2 subunits of the endocytic AP-2 adaptor coordinate proper assembly and operation of endocytic components during clathrin-mediated endocytosis. The 2 subunit appendage contains a common binding site for -arrestin or ARH. To determine the importance of this interaction surface in living cells, we used siRNA-based gene silencing. The effect of extinguishing 2-subunit expression on the internalization of transferrin is considerably weaker than an AP-2 -subunit knockdown. We show the mild sorting defect is due to fortuitous substitution of the 2 chain with the closely related endogenous 1 subunit of the AP-1 adaptor complex. Simultaneous silencing of both 1- and 2-subunit transcripts recapitulates the strong subunit RNAi phenotype, and results in loss of ARH from endocytic clathrin coats. An RNAi-insensitive 2-YFP expressed in the 1+2-silenced background restores cellular AP-2 levels, robust transferrin internalization, and ARH colocalization with cell surface clathrin. The importance of the -appendage platform subdomain over clathrin for precise deposition of ARH at clathrin assembly zones is revealed by a 2-YFP with a disrupted ARH binding interface, which does not restore ARH colocalization with clathrin. We also show a -arrestin 1 mutant, which engages coated structures in the absence of any GPCR stimulation, colocalizes with 2-YFP and clathrin even in the absence of an operational clathrin-binding sequence. These findings argue against ARH and -arrestin binding to a site upon the 2 appendage platform that is later obstructed by polymerized clathrin. We conclude that ARH and -arrestin depend on a privileged 2 appendage site for proper cargo recruitment to clathrin bud sites.
These authors contributed equally to this work.
Present address: Department of Medicine and HHMI, Washington University School of Medicine, St. Louis, Missouri 63110.
Address correspondence to:
Linton M. Traub (traub{at}pitt.edu)
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  J. R. Thieman, S. K. Mishra, K. Ling, B. Doray, R. A. Anderson, and L. M. Traub Clathrin Regulates the Association of PIPKI{gamma}661 with the AP-2 Adaptor {beta}2 Appendage J. Biol. Chem., May 15, 2009; 284(20): 13924 - 13939. [Abstract] [Full Text] [PDF]
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