Specific membrane binding of the carboxypeptidase Y inhibitor I(C), 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 phosphatidylethanolamine-binding protein family. The binding of I(C) 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-phosphate, phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate, with K(D) values below 100 nm. The liposome-binding assay and surface plasmon resonance analyses of I(C), when complexed with carboxypeptidase Y, and the mutant forms of I(C) 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 I(C) to cellular membranes was subsequently analyzed by fluorescence microscopy of yeast cells producing the green fluorescent protein-tagged I(C), suggesting that I(C) is specifically targeted to vacuolar membranes rather than cytoplasmic membranes, during the stationary growth phase. The present findings provide novel insights into the membrane-targeting and biological functions of I(C) and phosphatidylethanolamine-binding proteins.
Mima
Joji
J
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan. mima@kais.kyoto-u.ac.jp
Fukada
Hiroaki
H
Nagayama
Mitsuru
M
Ueda
Mitsuyoshi
M
D001665:Binding Sites; D002352:Carrier Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D049488:Phosphatidylethanolamine Binding Protein / Q000737:chemistry* / Q000378:metabolism*; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D020349:Surface Plasmon Resonance
D002352:Carrier Proteins; D049488:Phosphatidylethanolamine Binding Protein; D010714:Phosphatidylethanolamines; D010743:Phospholipids; D029701:Saccharomyces cerevisiae Proteins; C496670:carboxypeptidase Y inhibitor IC, S cerevisiae; D049452:Green Fluorescent Proteins; C483858:phosphatidylethanolamine