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Vol. 18, Issue 8, 2893-2903, August 2007

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Interaction of the Endocytic Scaffold Protein Pan1 with the Type I Myosins Contributes to the Late Stages of Endocytosis

Sarah L. Barker^*,, Linda Lee, B. Daniel Pierce^*, Lymarie Maldonado-Báez^*, David G. Drubin, and Beverly Wendland^*

*Department of Biology, The Johns Hopkins University, Baltimore, MD 21218; and Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720

Submitted May 11, 2007; Accepted May 11, 2007
Monitoring Editor: Sandra Schmid

The yeast endocytic scaffold Pan1 contains an uncharacterized proline-rich domain (PRD) at its carboxy (C)-terminus. We report that the pan1-20 temperature-sensitive allele has a disrupted PRD due to a frame-shift mutation in the open reading frame of the domain. To reveal redundantly masked functions of the PRD, synthetic genetic array screens with a pan1PRD strain found genetic interactions with alleles of ACT1, LAS17 and a deletion of SLA1. Through a yeast two-hybrid screen, the Src homology 3 domains of the type I myosins, Myo3 and Myo5, were identified as binding partners for the C-terminus of Pan1. In vitro and in vivo assays validated this interaction. The relative timing of recruitment of Pan1-green fluorescent protein (GFP) and Myo3/5-red fluorescent protein (RFP) at nascent endocytic sites was revealed by two-color real-time fluorescence microscopy; the type I myosins join Pan1 at cortical patches at a late stage of internalization, preceding the inward movement of Pan1 and its disassembly. In cells lacking the Pan1 PRD, we observed an increased lifetime of Myo5-GFP at the cortex. Finally, Pan1 PRD enhanced the actin polymerization activity of Myo5–Vrp1 complexes in vitro. We propose that Pan1 and the type I myosins interactions promote an actin activity important at a late stage in endocytic internalization.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Present address: Banting and Best Department of Medical Research, Terence Donnelly Centre for Cellular and Biomolecular Research, Toronto, Ontario M5S 3E1 Canada.

Address correspondence to: Beverly Wendland (bwendland{at}jhu.edu).

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