title: SLAC, a complex between Sla1 and Las17, regulates actin polymerization during clathrin-mediated endocytosis.
abstract: During clathrin-mediated endocytosis, branched actin polymerization nucleated by the Arp2/3 complex provides force needed to drive vesicle internalization. Las17 (yeast WASp) is the strongest activator of the Arp2/3 complex in yeast cells; it is not autoinhibited and arrives to endocytic sites 20 s before actin polymerization begins. It is unclear how Las17 is kept inactive for 20 s at endocytic sites, thus restricting actin polymerization to late stages of endocytosis. In this paper, we demonstrate that Las17 is part of a large and biochemically stable complex with Sla1, a clathrin adaptor that inhibits Las17 activity. The interaction is direct, multivalent, and strong, and was mapped to novel Las17 polyproline motifs that are simultaneously class I and class II. In vitro pyrene-actin polymerization assays established that Sla1 inhibition of Las17 activity depends on the class I/II Las17 polyproline motifs and is based on competition between Sla1 and monomeric actin for binding to Las17. Furthermore, live-cell imaging showed the interaction with Sla1 is important for normal Las17 recruitment to endocytic sites, inhibition during the initial 20 s, and efficient endocytosis. These results advance our understanding of the regulation of actin polymerization in endocytosis.
authors.affiliation: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Feliciano
authors.forename: Santiago M
authors.initials: SM
authors.lastname: Di Pietro
mesh_terms: D000199:Actins / Q000378:metabolism*; D020816:Amino Acid Motifs; D002966:Clathrin / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000378:metabolism*; D004705:Endocytosis; D013058:Mass Spectrometry; D046912:Multiprotein Complexes / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D058105:Polymerization; D011485:Protein Binding; D055550:Protein Stability; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D020798:Two-Hybrid System Techniques; D051316:Wiskott-Aldrich Syndrome Protein / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018909:src Homology Domains
chemical_list: D000199:Actins; D002966:Clathrin; D003598:Cytoskeletal Proteins; C480361:LAS17 protein, S cerevisiae; D046912:Multiprotein Complexes; C082265:SLA1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D051316:Wiskott-Aldrich Syndrome Protein