Distinct role of TGN-resident clathrin adaptors for Vps21p activation in the TGN-endosome trafficking pathway.

Clathrin-mediated vesicle trafficking plays central roles in post-Golgi transport. In yeast (Saccharomyces cerevisiae), the AP-1 complex and GGA adaptors are predicted to generate distinct transport vesicles at the trans-Golgi network (TGN), and the epsin-related proteins Ent3p and Ent5p (collectively Ent3p/5p) act as accessories for these adaptors. Recently, we showed that vesicle transport from the TGN is crucial for yeast Rab5 (Vps21p)-mediated endosome formation, and that Ent3p/5p are crucial for this process, whereas AP-1 and GGA adaptors are dispensable. However, these observations were incompatible with previous studies showing that these adaptors are required for Ent3p/5p recruitment to the TGN, and thus the overall mechanism responsible for regulation of Vps21p activity remains ambiguous. Here, we investigated the functional relationships between clathrin adaptors in post-Golgi-mediated Vps21p activation. We show that AP-1 disruption in the ent3Δ5Δ mutant impaired transport of the Vps21p guanine nucleotide exchange factor Vps9p transport to the Vps21p compartment and severely reduced Vps21p activity. Additionally, GGA adaptors, the phosphatidylinositol-4-kinase Pik1p and Rab11 GTPases Ypt31p and Ypt32p were found to have partially overlapping functions for recruitment of AP-1 and Ent3p/5p to the TGN. These findings suggest a distinct role of clathrin adaptors for Vps21p activation in the TGN-endosome trafficking pathway.

Distinct roles for plasma membrane PtdIns(4)P and PtdIns(4,5)P2 during receptor-mediated endocytosis in yeast.

Clathrin-mediated endocytosis requires the coordinated assembly of various endocytic proteins and lipids at the plasma membrane. Accumulating evidence demonstrates a crucial role for phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] in endocytosis but specific roles for phosphatidylinositol-4-phosphate [PtdIns(4)P], other than as the biosynthetic precursor of PtdIns(4,5)P2, have not been clarified. In this study we investigated the roles of PtdIns(4)P and PtdIns(4,5)P2 in receptor-mediated endocytosis through the construction of temperature-sensitive (ts) mutants for the phosphatidylinositol 4-kinases (PI4-kinases) Stt4p and Pik1p and the 1-phosphatidylinositol-4-phosphate 5-kinase [PtdIns(4) 5-kinase] Mss4p. Quantitative analyses of endocytosis revealed that both the stt4tspik1ts and mss4ts mutants have a severe defect in endocytic internalization. Live-cell imaging of endocytic protein dynamics in stt4tspik1ts and mss4ts mutants revealed that PtdIns(4)P is required for the recruitment of the α-factor receptor Ste2p to clathrin-coated pits, whereas PtdIns(4,5)P2 is required for membrane internalization. We also found that the localization to endocytic sites of the ENTH/ANTH domain-bearing clathrin adaptors, Ent1p, Ent2p, Yap1801p and Yap1802p, is significantly impaired in the stt4tspik1ts mutant but not in the mss4ts mutant. These results suggest distinct roles in successive steps for PtdIns(4)P and PtdIns(4,5)P2 during receptor-mediated endocytosis.