The Function of the Endocytic Scaffold Protein Pan1p Depends on Multiple Domains

Abstract

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...

Interaction of the Endocytic Scaffold Protein Pan1 with the Type I Myosins Contributes to the Late Stages of Endocytosis

“…Consistent with this idea, pan1-20 cells that also lack the key PRD region show exaggerated actin structures that seem to be either very thick cables or dramatically elongated cortical patches when grown at the nonpermissive temperature (Wendland et al, 1996). Alternatively, the pan1-20 and pan1⌬PRD proteins may have other activities affected in addition to their altered myosin SH3 ligand, such as putative regulated interactions between the N-termini and C-termini of Pan1 (Miliaras et al, 2004;Toshima et al, 2005).…”

“…However, the PRD of PAN1 is not normally essential to cells, because previous studies have failed to observe any fitness defect or temperature sensitivity when the PRD was deleted (Sachs and Deardorff, 1992;Duncan et al, 2001;Miliaras et al, 2004). It is possible that the temperature-sensitive and endocytic defects of the pan1-20 mutation might be due to more than the loss of the Cterminal sequence within the PRD, perhaps through a subtle effect on protein stability or the additional nonnative Cterminal residues (Figure 2A).…”

“…The later events in internalization, possibly including vesicle scission, involve actin polymerization (Yarar et al, 2005) and Arp2/3 complex recruitment (Merrifield et al, 2005). The central region of Pan1 mediates the formation of Pan1-Pan1 complexes (Miliaras et al, 2004), and also seems to link the N-terminal and C-terminal functions of Pan1 into a single protein complex (Miliaras et al, 2004;Toshima et al, 2005). Thus, Pan1 is poised to coordinate and regulate the sequence of early-to-late events necessary for efficient endocytic internalization.…”

Wsp1 Is Downstream of Cin1 and Regulates Vesicle Transport and Actin Cytoskeleton as an Effector of Cdc42 and Rac1 in Cryptococcus neoformans

“…The EH domains bind the SlaI, End3, Ent1/2, and Yap1801/2 proteins (5, 43, 50), while the A domain interacts with the Arp2/3 complex. Pan1 does not interact with Las17/Bee1, which lacks the GBD, but interacts through the inhibitory protein SlaI to activate the Arp2/3 complex (13,27,42,50).…”

Yeast P4-ATPases Drs2p and Dnf1p Are Essential Cargos of the NPFXD/Sla1p Endocytic Pathway

“…Therefore, Pan1p has the capacity to link adaptor-bound cargo proteins to clathrincoated pits and sites of actin assembly. Pan1p, End3p, and actin assembly are required for both ubiquitin (Ub)-dependent and NPFXD-dependent endocytosis, although Sla1p is only required for endocytosis of cargo bearing the NPFXD signal (Howard et al, 2002;Miliaras et al, 2004). Drs2p not only has the potential to physically interact with the Sla1p/ Pan1p/End3p complex but also it is functionally linked to this complex as drs2⌬ is synthetically lethal with the temperature-conditional pan1-20 allele (Chen et al, 1999).…”