One our laboratory's long-term goals is to understand how clathrin light chain regulates the endocytic process. This proposal is to study LC in the Saccharomyces Cerevisiae model system, which provides the ability to knockout, replace or otherwise alter genes of interest and the availability of screens for interacting molecules or intersecting pathways. It has long been known that deletion of either the heavy (HC) or light (LC) chains of clathrin in yeast caused defects in a-factor uptake and endocytic patch progression. Our lab was one of the first to recognize that LC could suppress the endocytic defects of a HC null. In addition we demonstrated that this suppression was mediated by the N-terminus (amino acids 1-143) of LC (LC-NT). Yeast two-hybrid using LC as bait identified the PIP2 binding, talin-like protein Sla2 (the yeast homologue of the mammalian endocytic adaptors Hip1/Hip1 R) as a novel binding partner and this interaction was further mapped to LC-NT. We believe that LC suppression of HC deletion involves the regulation of Sla2 by LC. As such, the main goal of this proposal is to identify how LC regulation of Sla2 promotes endocytic progression from cortical patches to vesicle formation. We will test two models for how LC regulates Sla2 interaction the actin cytoskeleton. In addition, we plan to screen for proteins which have functional redundancy with the LC-NT, which will provide us with a more complete picture of regulation in this system. Endocytic dysfunction can contribute to a diverse list of diseases which pose threats to public health in America today, including: hypercholesterolemia, neurological disorders and tumorigenesis. Although mammalian LC is well studied, nearly all functional data pertaining to this protein was assessed in vitro. Since many of the mammalian proteins involved in endocytosis, including clathrin, are conserved in yeast it makes sense to take advantage of this model system, which allows us to analyze proteins in vivo. This proposal will examine the in vivo regulation of Sla2 by LC, and provide important observations, which can then be applied to mammalian systems. As such our studies may reveal new information concerning this vital process and eventually help produce new strategies for management of these diseases. ? ? ?
