Discovery and characterization of the SLAC complex and its role in actin polymerization during clathrin-mediated endocytosis
Date
2013
Authors
Feliciano, Daniel, author
Di Pietro, Santiago, advisor
Bamburg, James, committee member
Curthoys, Norman, committee member
Chen, Chaoping, committee member
Reist, Noreen, committee member
Journal Title
Journal ISSN
Volume Title
Abstract
Endocytosis is the process by which cells control the lipid and transmembrane protein compositions in order to comply with certain requirements essential for cellular function. The different forms of endocytosis provide the cell with a discriminatory system where specific cargoes are selected, packed and internalized when there is a particular physiological demand. Given the importance of endocytic internalization routes for a variety of cellular processes, it is not surprising that defects in the protein machinery involved in these pathways leads to pathologies. Examples of some metabolic disorders associated to defects in adaptor or receptor function include autosomal recessive and familial hypercholesterolemia. In other cases, mutations in actin regulatory proteins, such as WASp, can cause many blood disorders that include primary immunodeficiency and thrombocytopenia. Clathrin-mediated endocytosis (CME) is a fundamental pathway conserved from yeast to humans that proceeds by forming a clathrin coat at the plasma membrane followed by the recruitment of proteins that promote membrane curvature, actin polymerization, and scission. CME is the mayor route for nutrient uptake, distribution of membrane components, and receptor internalization. During CME, branched actin polymerization nucleated by the Arp2/3 complex provides force needed to drive vesicle internalization. Las17 (WASp) is the strongest activator of the Arp2/3 complex in yeast cells, it is not autoinhibited, and arrives to endocytic sites 20 seconds before actin polymerization begins. One of the most outstanding questions in the field has been how Las17 is inhibited during the initial 20 seconds after its arrival to sites of endocytosis. In this dissertation, the discovery and characterization of a stable complex between Las17 and the clathrin adaptor Sla1 is described, in which Las17 is inhibited. This interaction is direct, multivalent, and strong, and was mapped to novel Las17 polyproline motifs that are simultaneously class I (RxxPxxP) and class II (PxxPxR). In vitro pyrene-actin polymerization assays established that Sla1 inhibition of Las17 activity depends on a new class I/II Las17 polyproline motifs. The inhibition is based on competition between Sla1 and monomeric actin for binding to sequences comprising a novel G-actin binding site in Las17 that is also characterized. The Las17 novel G-actin binding module 1 (LGM1) requires two sets of arginine-rich sites for normal Las17 function in vitro and in vivo. Furthermore, live cell imaging showed the interaction with Sla1 is important for normal Las17 recruitment to endocytic sites, its inhibition during the initial 20 seconds, and for efficient endocytosis. Within this complex, Las17 requires full length Bzz1, a membrane tubulation protein, for its activation in vitro through a mechanism that does not depend on complex dissociation. Since Sla1 and Las17 regulate actin polymerization during clathrin-mediated endocytosis, this complex has been named SLAC. The discovery and characterization of the SLAC complex help to define the negative and positive mechanisms regulating Las17 activity and answer one of the most outstanding questions in the field. This work also sets the stage to decipher the roles of other WASp homologues in mammalian cells. Overall, findings reported here advance our understanding of the regulation of actin polymerization by Las17 during clathrin-mediated endocytosis.
Description
Rights Access
Subject
cctin
WASp
Las17
endocytosis
clathrin