Early-Arriving Syp1p and Ede1p Function in Endocytic Site Placement and Formation in Budding Yeast

Abstract

Recent studies have revealed the detailed timing of protein recruitment to endocytic sites in budding yeast. However, little is understood about the early stages of their formation. Here we identify the septin-associated protein Syp1p as a component of the machinery that drives clathrin-mediated endocytosis in budding yeast. Syp1p arrives at endocytic sites early in their formation and shares unique dynamics with the EH-domain protein Ede1p. We find that Syp1p is related in amino acid sequence to several mammalian proteins one of which, SGIP1-␣, is an endocytic component that binds the Ede1p homolog Eps15. Like Syp1p, SGIP1-␣ arrives early at sites of clathrin-mediated endocytosis, suggesting that Syp1p/Ede1p and SGIP1-␣/Eps15 may have a conserved function. In yeast, both Syp1p and Ede1p play important roles in the rate of endocytic site turnover. Additionally, Ede1p is important for endocytic site formation, whereas Syp1p acts as a polarized factor that recruits both Ede1p and endocytic sites to the necks of emerging buds. Thus Ede1p and Syp1p are conserved, early-arriving endocytic proteins with roles in the formation and placement of endocytic sites, respectively. INTRODUCTIONThe dynamics of protein recruitment to sites of clathrinmediated endocytosis have been revealed by live-cell microscopy in budding yeast and mammalian cells (Merrifield et al., 2002;Kaksonen et al., 2003;Kaksonen et al., 2005). These studies have identified numerous proteins that sequentially assemble at endocytic sites and have shown that actin polymerization can power internalization. It is now evident that the dynamic recruitment and disappearance of endocytic proteins are precisely coordinated for productive internalization and that each protein has defined dynamics at endocytic sites. In Saccharomyces cerevisiae four endocytic modules have been defined that each contain proteins with similar dynamics: the coat, WASP/myo, amphiphysin, and actin modules (Kaksonen et al., 2005).Despite detailed knowledge of events at endocytic sites, little is understood about the early stages of their formation. The best candidates for proteins that initiate endocytic site formation are those that arrive earliest. In mammalian cells the classical coat protein clathrin marks the earliest known stage of endocytic site formation (Merrifield et al., 2002). Additionally, the adapter AP-2, is critical for site formation and has similar dynamic behavior to clathrin and so is thought to arrive early (Hinrichsen et al., 2003;Motley et al., 2003;Ehrlich et al., 2004;Keyel et al., 2004). In yeast, the role of AP-2 is unclear, but clathrin, a coat module component, marks the first stages of endocytosis, and its deletion causes severe defects in the number of sites formed (Huang et al., 1999;Kaksonen et al., 2005;Newpher et al., 2005;Newpher and Lemmon, 2006). An additional yeast protein, Ede1p, arrives early and plays a role in endocytic site formation, although its dynamics and function have yet to be fully investigated (Kaksonen et al., 2005;Toshima et al., 200...

Morphogenesis and the Cell Cycle

“…In some cases, direct mechanistic links have been identified: septin rings recruit the formin Bnr1p, which nucleates actin cable formation in mother cells (Pruyne et al 2004a). Septins also recruit the endocytic actin patch initiator protein Syp1p, promoting patch clustering at the mother-bud neck (Qiu et al 2008;Stimpson et al 2009). In other cases, the evidence is less direct.…”

Quantifying the dynamic interactions between a clathrin-coated pit and cargo molecules

“…In addition, the number of fluorescently labeled clathrin molecules incorporated into a pit is burdened by Poisson statistics, making it very difficult to reliably characterize the growth rate at the single CCP level. Nevertheless, it is possible to use the ensemble average to gather statistical information on the growth (17,18). The second problem encountered with this approach is that a CCP is not flat on the cell membrane.…”

Regulation of ubiquitin-dependent cargo sorting by multiple endocytic adaptors at the plasma membrane

“…When overexpressed, the FCHO proteins, Eps15, and intersectin may precede the arrival of AP-2 and clathrin on the plasma membrane, although all of these factors exhibit highly similar recruitment kinetics (7,9,10). Current models suggest that the FCHO proteins initiate membrane bending and cargo clustering via their FCH-BAR (F-BAR) and μ-homology domains, respectively (7,8,11).…”