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.
Miliaras
Nicholas B
NB
Department of Biology, The Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA.
Park
Jin-Hyouk
JH
Wendland
Beverly
B
D000199:Actins / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology* / Q000633:toxicity; D017930:Genes, Reporter; D008840:Microfilament Proteins; D010446:Peptide Fragments / Q000235:genetics / Q000502:physiology; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D051964:Receptors, Mating Factor; D043563:Receptors, Pheromone / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism
D000199:Actins; D005656:Fungal Proteins; D008840:Microfilament Proteins; C111870:PAN1 protein, S cerevisiae; D010446:Peptide Fragments; D043562:Receptors, G-Protein-Coupled; D051964:Receptors, Mating Factor; D043563:Receptors, Pheromone; C081863:STE3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C061570:protein kinase N; D020794:Receptor Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C