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

  • Wataru Yamamoto
    Tokyo University of Science 1 Department of Biological Science and Technology , , 6-3-1 Niijyuku, Katsushika-ku, Tokyo 125-8585 , Japan
  • Suguru Wada
    Tokyo University of Science 1 Department of Biological Science and Technology , , 6-3-1 Niijyuku, Katsushika-ku, Tokyo 125-8585 , Japan
  • Makoto Nagano
    Tokyo University of Science 1 Department of Biological Science and Technology , , 6-3-1 Niijyuku, Katsushika-ku, Tokyo 125-8585 , Japan
  • Kaito Aoshima
    Tokyo University of Science 1 Department of Biological Science and Technology , , 6-3-1 Niijyuku, Katsushika-ku, Tokyo 125-8585 , Japan
  • Daria Elisabeth Siekhaus
    Institute of Science and Technology Austria 2 , Am Campus 1, A-3400 Klosterneuburg , Austria
  • Junko Y. Toshima
    Tokyo University of Technology 3 School of Health Science , , 5-23-22 Nishikamata, Ota-ku, Tokyo 144-8535 , Japan
  • Jiro Toshima
    Tokyo University of Science 1 Department of Biological Science and Technology , , 6-3-1 Niijyuku, Katsushika-ku, Tokyo 125-8585 , Japan

Abstract

<jats:title>ABSTRACT</jats:title> <jats:p>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 stt4ts pik1ts and mss4ts mutants have a severe defect in endocytic internalization. Live-cell imaging of endocytic protein dynamics in stt4ts pik1ts 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 stt4ts pik1ts 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.</jats:p>

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