tailieunhanh - Báo cáo khoa học: Specific membrane binding of the carboxypeptidase Y inhibitor IC, a phosphatidylethanolamine-binding protein family member

I C , an endogenous cytoplasmic inhibitor of vacuolar carboxypeptidase Y in the yeast Saccharomyces cerevisiae, is classified as a member of the phos-phatidylethanolamine-binding protein family. The binding of I C to phos-pholipid membranes was first analyzed using a liposome-binding assay and by surface plasmon resonance measurements, which revealed that the affin- | ễFEBS Journal Specific membrane binding of the carboxypeptidase Y inhibitor IC a phosphatidylethanolamine-binding protein family member Joji Mima Hiroaki Fukada Mitsuru Nagayama and Mitsuyoshi Ueda Division of Applied Life Sciences Graduate Schoolof Agriculture Kyoto University Japan Keywords IC membrane binding PEBP phosphatidylserine phosphoinositide Correspondence J. Mima Division of Applied Life Sciences Graduate Schoolof Agriculture Kyoto University Kitashirakawa Sakyo-ku Kyoto 606-8502 Japan Fax 81 75 753 6112 Tel 81 75 753 6125 E-mail mima@ Present address Department of Biochemistry Dartmouth Medical School Hanover NH USA Received 7 July 2006 revised 4 October 2006 accepted 9 October 2006 doi IC an endogenous cytoplasmic inhibitor of vacuolar carboxypeptidase Y in the yeast Saccharomyces cerevisiae is classified as a member of the phosphatidylethanolamine-binding protein family. The binding of IC to phospholipid membranes was first analyzed using a liposome-binding assay and by surface plasmon resonance measurements which revealed that the affinity of this inhibitor was not for phosphatidylethanolamine but for anionic phospholipids such as phosphatidylserine phosphatidylinositol 3-phos-phate phosphatidylinositol 3 4-bisphosphate and phosphatidylinositol 3 4 5-trisphosphate with KD values below 100 nM. The liposome-binding assay and surface plasmon resonance analyses of IC when complexed with carboxypeptidase Y and the mutant forms of IC further suggest that the N-terminal segment Met1-His18 in its carboxypeptidase Y-binding sites is involved in the specific and efficient binding to anionic phospholipid membranes. The binding of IC to cellular membranes was subsequently analyzed by fluorescence microscopy of yeast cells producing the green fluorescent protein-tagged IC suggesting that IC is specifically targeted to vacuolar membranes rather than cytoplasmic membranes during the stationary growth phase. The .