A rapid cascade of regulatory events defines the developmental fates of embryonic cells. However, once established, these developmental fates and the underlying transcriptional programs can be remarkably stable. Here, we describe two proteins, MEP-1 and LET-418/Mi-2, required for maintenance of somatic differentiation in C. elegans. In animals lacking MEP-1 and LET-418, germline-specific genes become derepressed in somatic cells, and Polycomb group (PcG) and SET domain-related proteins promote this ectopic expression. MEP-1 and LET-418 interact in vivo with the germline-protein PIE-1. Our findings support a model in which PIE-1 inhibits MEP-1 and associated factors to maintain the pluripotency of germ cells, while at later times MEP-1 and LET-418 remodel chromatin to establish new stage- or cell-type-specific differentiation potential.

The role of aggregation of abnormal proteins in cellular toxicity is of general importance for understanding many neurological disorders. Here, using a yeast model, we demonstrate that mutations in many proteins involved in endocytosis and actin function dramatically enhance the toxic effect of polypeptides with an expanded polyglutamine (polyQ) domain. This enhanced cytotoxicity required polyQ aggregation and was dependent on the yeast protein Rnq1 in its prion form. In wild-type cells, expression of expanded polyQ followed by its aggregation led to specific and acute inhibition of endocytosis, which preceded growth inhibition. Some components of the endocytic machinery were efficiently recruited into the polyQ aggregates. Furthermore, in cells with polyQ aggregates, cortical actin patches were delocalized and actin was recruited into the polyQ aggregates. Aggregation of polyQ in mammalian HEK293 cells also led to defects in endocytosis. Therefore, it appears that inhibition of endocytosis is a direct consequence of polyQ aggregation and could significantly contribute to cytotoxicity.

Endocytosis is a protein and lipid-trafficking pathway that occurs in all eukaryotic cells. It involves the internalization of plasma membrane proteins and lipids into the cell and the subsequent degradation of proteins in the lysosome or the recycling of proteins and lipids back to the plasma membrane. Over the past decade, studies in yeast and mammalian cells have revealed endocytosis to be a very complex molecular process that depends on regulated interactions between a variety of proteins and lipids. The Eps15 homology (EH) domain is a conserved, modular protein-interaction domain found in several endocytosis proteins. EH proteins can function as key regulators of endocytosis through their ability to interact with many of the other proteins involved in this process.

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...

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