Interaction of the Endocytic scaffold protein Pan1 with the type I Myosins contributes to the late stages of endocytosis

被引:33
作者
Barker, Sarah L.
Lee, Linda
Pierce, B. Daniel
Maldonado-Baez, Lymarie
Drubin, David G.
Wendland, Beverly [1 ]
机构
[1] Johns Hopkins Univ, Dept Biol, Baltimore, MD 21218 USA
[2] Univ Calif Berkeley, Dept Mol & Cell Biol, Berkeley, CA 94720 USA
[3] Terence Donnelly Ctr Cellular & Biomol Res, Dept Med Res, Toronto, ON M5S 3E1, Canada
关键词
D O I
10.1091/mbc.E07-05-0436
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
The yeast endocytic scaffold Pan1 contains an uncharacterized proline-rich domain (PRD) at its carboxy (Q-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 pan1 Delta PRD 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.
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页码:2893 / 2903
页数:11
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