Pan1p is an essential protein of the yeast Saccharomyces cerevisiae that is required for the internalization step of endocytosis and organization of the actin cytoskeleton. Pan1p, which binds several other endocytic proteins, is composed of multiple protein-protein interaction domains including two Eps15 Homology (EH) domains, a coiled-coil domain, an acidic Arp2/3-activating region, and a prolinerich domain. In this study, we have induced high-level expression of various domains of Pan1p in wild-type cells to assess the dominant consequences on viability, endocytosis, and actin organization. We found that the most severe phenotypes, with blocked endocytosis and aggregated actin, required expression of nearly full length Pan1p, and also required the endocytic regulatory protein kinase Prk1p. The central coiled-coil domain was the smallest fragment whose overexpression caused any dominant effects; these effects were more pronounced by inclusion of the second EH domain. Co-overexpressing nonoverlapping amino-and carboxy-terminal fragments did not mimic the effects of the intact protein, whereas fragments that overlapped within the coiled-coil region could. Yeast two-hybrid and in vivo coimmunoprecipitation analyses suggest that Pan1 may form dimers or higher order oligomers. Collectively, our data support a view of Pan1p as a dimeric/oligomeric scaffold whose functions require both the amino-and carboxy-termini, linked by the central region.Key words: actin, EH domain, End3p, endocytosis, Pan1p, Prk1p, proline-rich domain, scaffold, yeast Endocytosis is a fundamental protein and lipid trafficking pathway in which plasma membrane proteins and lipids are internalized into vesicles and then delivered to early endosomes. Subsequently, receptor proteins may be either recycled back to the plasma membrane or sent on to late endosomes and lysosomes for degradation. Endocytosis is a complex molecular process involving clathrin and adaptor proteins, the actin cytoskeleton, ubiquitin, and various protein and lipid kinases and phosphatases (reviewed in (1,2)). Furthermore, this complexity seems to be functionally conserved throughout eukaryotes, as many endocytic proteins that have been identified through genetic studies in yeast have homologues in mammalian cells.Pan1p is a conserved, essential protein in Saccharomyces cerevisiae, which functions in actin polarization, cytokinesis (3) and endocytosis (4). It is a large, modular protein consisting of two Eps15 homology (EH) domains within its aminoterminal region (5), a central coiled-coil region, an acidic region which binds to and activates the Arp2/3 complex (6), and a proline-rich region at its carboxy-terminus. The EH domain is a conserved protein binding motif of approximately 70 amino acids that is found in many endocytic proteins (7). These include the mammalian Pan1p-related proteins Eps15 (8) and intersectin (9), and the yeast proteins End3p (10) and Ede1p (11). The second EH domain of Pan1p binds the yeast epsins Ent1p and Ent2p and the yeast homologues of a mammal...