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Volume 270, Number 16, Issue of April 21, pp. 9147-9153, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.

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Adisak Wongkajornsilp , Terrone L. Rosenberry

A HeLa S3 subline is unusual in accumulating relatively large amounts of glucosaminyl(acyl)phosphatidylinositol (GlcN(acyl)PI), a derivative of phosphatidylinositol (PI) in which both GlcN and a fatty acid are linked to inositol hydroxyl groups (D. Sevlever, D. Humphrey, and T. L. Rosenberry, submitted for publication). This lipid is a proposed intermediate on the biosynthetic pathway for glycosyl-PI (GPI) anchors of membrane proteins. In this study we demonstrate for the first time that exogenous inositol phospholipids can enter this biosynthetic pathway and be metabolized to GlcN(acyl)PI. When HeLa S3 cells were incubated for 24 h with exogenous PI or sn-1-acyl-2- lyso-phosphatidylinositol (lyso-PI) labeled with H in the inositol group, 25-30% of the label was recovered in cell-associated lipids and most of the remaining 70-75% in hydrophilic metabolites in the medium. The predominant labeled lipid was PI, with smaller amounts of lyso-PI, phosphatidylinositol 4-phosphate (PIP), and GlcN(acyl)PI. Both exogenous lipid precursors gave the same distribution of labeled lipids, and a similar distribution was observed for endogenous inositol phospholipids metabolically labeled with [H]inositol. Addition of excess inositol had no effect on the conversion of [H]lyso-PI to [H]GlcN(acyl)PI, indicating that the conversion did not result from breakdown to [H]inositol followed by resynthesis. The cellular orientation of incorporated PI and lyso-PI was determined by incubating cells at 4 °C with PI-specific phospholipase C (PI-PLC). This enzyme cleaves only PI and lyso-PI on the outer leaflet of the cell membrane. After 24-h incubation with either precursor, only about 15% of cell-associated [H]PI or [H]lyso-PI was on the outer leaflet. However, more than 60% of the [H]PI was on the outer leaflet after 1-h incubation with either precursor, suggesting that substantial sn-2 acylation of exogenous [H]lyso-PI occurred in the outer leaflet. This suggestion was confirmed by examining labeled lipids in cells after uptake of [H]lyso-PI at 4 °C. No transmembrane translocation of lyso-PI, PI phosphorylation, or PI glycosylation occurred at this temperature, but some sn-2 acylation was apparent and more than 90% of the [H]PI formed was on the outer leaflet. These data indicate that sn-2 acylation can occur in the outer leaflet of the cell membrane, perhaps by transacylation from other cell surface phospholipids.

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