Centrioles: some self-assembly required.

Centrioles play an important role in organizing microtubules and are precisely duplicated once per cell cycle. New (daughter) centrioles typically arise in association with existing (mother) centrioles (canonical assembly), suggesting that mother centrioles direct the formation of daughter centrioles. However, under certain circumstances, centrioles can also selfassemble free of an existing centriole (de novo assembly). Recent work indicates that the canonical and de novo pathways utilize a common mechanism and that a mother centriole spatially constrains the self-assembly process to occur within its immediate vicinity. Other recently identified mechanisms further regulate canonical assembly so that during each cell cycle, one and only one daughter centriole is assembled per mother centriole.

The function of the endocytic scaffold protein Pan1p depends on multiple domains.

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 proline-rich 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.

EH proteins: multivalent regulators of endocytosis (and other pathways).

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.

Aggregation of expanded polyglutamine domain in yeast leads to defects in endocytosis.

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.