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

Abstract

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 stt4^tspik1^ts and mss4^ts mutants have a severe defect in endocytic internalization. Live-cell imaging of endocytic protein dynamics in stt4^tspik1^ts and mss4^ts 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 stt4^tspik1^ts mutant but not in the mss4^ts mutant. These results suggest distinct roles in successive steps for PtdIns(4)P and PtdIns(4,5)P₂ during receptor-mediated endocytosis.

Keywords: Clathrin; ENTH/ANTH; Endocytosis; PI(4)P; PI(4,5)P2.