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...

ETOC: During yeast endocytic site formation, Ede1p (yeast Eps15), but not clathrin light chain, is important for the recruitment of most other early-arriving proteins to endocytic sites. Cargo and clathrin light chain may play roles in regulating the transition of endocytic sites out of the “intermediate coat” stage of endocytosis.

Summary Like Ricin, Shiga, and Cholera toxins, yeast K28 is an A/B toxin that depends on endocytosis and retrograde trafficking for toxicity. Knowledge of the specific proteins, lipids, and mechanisms required for trafficking and killing by these toxins remains incomplete. Since K28 is a model for clinically relevant toxins, we screened over 5000 yeast mutants, identifying 365 that affect K28 sensitivity. Hypersensitive mutants revealed cytoprotective pathways, including stress-activated signaling and protein degradation. Resistant mutants clustered to endocytic, lipid organization and cell wall biogenesis pathways. Furthermore, GPI anchors and transcriptional regulation are important for K28-cell binding. Strikingly, the AP2 complex, which in metazoans links endocytic cargo to the clathrin coat, but had no assigned function in yeast, was critical for K28 toxicity. Yeast AP2 localizes to endocytic sites and has a cargo-specific function in K28 uptake. This comprehensive genetic analysis identified conserved processes important for A/B toxin trafficking and killing.

Plasma membrane transporters are often downregulated by their substrates. The yeast manganese transporter Smf1 is subject to two levels of regulation: heavy metals induce its sequestration within the cell, and also its ubiquitination and degradation in the vacuole. Degradation requires Bsd2, a membrane protein with a PPxY motif that recruits the ubiquitin ligase Rsp5, and which has a role in the quality control of membrane proteins, that expose hydrophilic residues to the lipid bilayer. We show that degradation of Smf1 requires in addition one of a pair of related yeast proteins, Tre1 and Tre2, that also contain PPxY motifs. Tre1 can partially inhibit manganese uptake without Bsd2, but requires Bsd2 to induce Smf1 degradation. It has a relatively hydrophilic transmembrane domain and binds to Bsd2. We propose that the Tre proteins specifically link Smf1 to the Bsd2-dependent quality control system. Their luminal domains are related to the transferrin receptor, but these are dispensable for Smf1 regulation. Tre proteins and the transferrin receptors appear to have evolved independently from the same family of membraneassociated proteases.