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Vol. 18, Issue 5, 1887-1896, May 2007

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The /1 and /2 Hemicomplexes of Clathrin Adaptors AP-1 and AP-2 Harbor the Dileucine Recognition Site

Balraj Doray^*,, Intaek Lee^*,, Jane Knisely, Guojun Bu, and Stuart Kornfeld^*

Departments of ^a*Internal Medicine and Pediatrics, Washington University School of Medicine, St. Louis, MO 63110

Submitted January 10, 2007; Revised February 22, 2007; Accepted March 1, 2007
Monitoring Editor: Sandra Schmid

The clathrin adaptors AP-1 and AP-2 bind cargo proteins via two types of motifs: tyrosine-based Yxx and dileucine-based [DE]XXXL[LI]. Although it is well established that Yxx motifs bind to the µ subunits of AP-1 or AP-2, dileucine motifs have been reported to bind to either the µ or subunits of these adaptors as well as the /1 hemicomplex of AP-1. To clarify this controversy, the various subunits of AP-1 and AP-2 were expressed individually and in hemicomplex form in insect cells, and they were used in glutathione S-transferase pull-down assays to determine their binding properties. We report that the /1 or /2 hemicomplexes bound the dileucine-based motifs of several proteins quite strongly, whereas binding by the 1/µ1 and 2/µ2 hemicomplexes, and the individual or µ subunits, was extremely weak or undetectable. The /1 and /2 hemicomplexes displayed substantial differences in their preference for particular dileucine-based motifs. Most strikingly, an aspartate at position –4 compromised binding to the /1 hemicomplex, whereas minimally affecting binding to /2. There was an excellent correlation between binding to the /2 hemicomplex and in vivo internalization mediated by the dileucine-based sorting signals. These findings provide new insights into the trafficking mechanisms of D/EXXXL[LI]-mediated sorting signals.

These authors contributed equally to this work.

Address correspondence to: Stuart Kornfeld (skornfel{at}im.wustl.edu)