PAN1P, YEAST EPS15, FUNCTIONS AS A MULTIVALENT ADAPTER THAT COORDINATES PROTEIN-PROTEIN INTERACTIONS ESSENTIAL FOR ENDOCYTOSIS

A genetic screen for factors required for endocytosis in the budding y east Saccharomyces cerevisiae previously identified PAN1. Pan1p is a h omologue of the mammalian protein eps15, which has been implicated in endocytosis by virtue of its association with the plasma membrane clat hrin adaptor complex AP-2. Pan1p contains two eps15 homology (EH) doma ins, a protein-protein interaction motif also present in other protein s that function in membrane trafficking. To address the role of Pan1p and EH domains in endocytosis, a yeast two-hybrid screen was performed using the EH domain-containing region of Pan1p, This screen identifie d yAP180A, one of two yeast homologues of a class of clathrin assembly proteins (AP1SO) that exhibit in vitro clathrin cage assembly activit y. In vitro binding studies using GST fusion proteins and yeast extrac ts defined distinct binding sites on yAP180A for Pan1p and clathrin. y AP180 proteins and Pan1p, like actin, localize to peripheral patches a long the plasma membrane. Mammalian synaptojanin, a phosphatidylinosit ol polyphosphate-5-phosphatase, also has been implicated in endocytosi s recently, and three synaptojanin-like genes have been identified in yeast. We observed genetic interactions between the yeast SJL1 gene an d PAN1, which suggest a role for phosphoinositide metabolites in Pan1p function. Together with other studies, these findings suggest that Pa n1p coordinates regulatory interactions between proteins required for both endocytosis and actin-cytoskeleton organization; these proteins i nclude the yAP180 proteins, clathrin, the ubiquitin-protein ligase Rsp 5p, End3p, and synaptojanin, We suggest that Pan1p (and by extension e ps15) serves as a multivalent adaptor around which dynamic interaction s between structural and regulatory components of the endocytic pathwa y converge.