Specific membrane binding of the carboxypeptidase Y inhibitor IC, a phosphatidylethanolamine‐binding protein family member

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‐phosphate, 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 present findings provide novel insights into the membrane‐targeting and biological functions of IC and phosphatidylethanolamine‐binding proteins.

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