Interaction of the endocytic scaffold protein Pan1 with the type I myosins contributes to the late stages of endocytosis.
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 pan1DeltaPRD 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.
Barker
Sarah L
SL
Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA.
Lee
Linda
L
Pierce
B Daniel
BD
Maldonado-Báez
Lymarie
L
Drubin
David G
DG
Wendland
Beverly
B
D051376:Actin-Related Protein 2-3 Complex / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D004705:Endocytosis; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D024461:Myosin Type I / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature; D020798:Two-Hybrid System Techniques; D018909:src Homology Domains
D051376:Actin-Related Protein 2-3 Complex; D000199:Actins; D005656:Fungal Proteins; D008840:Microfilament Proteins; C111870:PAN1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D024461:Myosin Type I