Distinct Actin and Lipid Binding Sites in Ysc84 Are Required during Early Stages of Yeast Endocytosis.

Agnieszka N Urbanek,Ellen G Allwood,Wesley I Booth,Adam P Smith,Kathryn R Ayscough,Robert Alan Arkowitz

doi:10.1371/journal.pone.0136732

Abstract

During endocytosis in S. cerevisiae, actin polymerization is proposed to provide the driving force for invagination against the effects of turgor pressure. In previous studies, Ysc84 was demonstrated to bind actin through a conserved N-terminal domain. However, full length Ysc84 could only bind actin when its C-terminal SH3 domain also bound to the yeast WASP homologue Las17. Live cell-imaging has revealed that Ysc84 localizes to endocytic sites after Las17/WASP but before other known actin binding proteins, suggesting it is likely to function at an early stage of membrane invagination. While there are homologues of Ysc84 in other organisms, including its human homologue SH3yl-1, little is known of its mode of interaction with actin or how this interaction affects actin filament dynamics. Here we identify key residues involved both in Ysc84 actin and lipid binding, and demonstrate that its actin binding activity is negatively regulated by PI(4,5)P2. Ysc84 mutants defective in their lipid or actin-binding interaction were characterized in vivo. The abilities of Ysc84 to bind Las17 through its C-terminal SH3 domain, or to actin and lipid through the N-terminal domain were all shown to be essential in order to rescue temperature sensitive growth in a strain requiring YSC84 expression. Live cell imaging in strains with fluorescently tagged endocytic reporter proteins revealed distinct phenotypes for the mutants indicating the importance of these interactions for regulating key stages of endocytosis.

Highlights

The actin cytoskeleton plays a central role in many dynamic cell processes including cell motility, cell organization and membrane trafficking
The full Ysc84 Actin Binding (YAB) domain has a high level of homology across eukaryotes, 43% identity between yeast and human, which is higher than most other actin binding proteins strongly suggesting a conserved function
Actin binding has been associated with the presence of basic amino acid residues and hydrophobic stretches incorporating leucine (L) [13], we analysed the amino acid sequence of the YAB domain for conserved residues of this nature

Summary

Introduction

The actin cytoskeleton plays a central role in many dynamic cell processes including cell motility, cell organization and membrane trafficking. Actin is highly conserved and forms dynamic filaments that can be regulated through binding myriad actin-binding proteins which control its organization and turnover in response to various external and internal stimuli [1]. The actin cytoskeleton is proposed to function both to provide the force required for plasma membrane invagination and subsequently during movement of the vesicle away from the plasma membrane [2, 3]. Endocytic Function of Ysc Actin and Lipid Binding

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