TEMPERATURE REGULATION OF THE TETRAHYMENA MIMBRES GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED PROTEIN-LIPID COMPOSITION

By incubating Tetrahymena mimbres cells with [H-3]myristic acid, [H-3] ethanolamine, [H-3]inositol, and [H-3]mannose, proteins having apparen t molecular masses of 23 and 63 kDa were identified as the cells' prin cipal glycosylphosphatidylinositol (GPI)-anchored proteins. These prot eins accounted for as much as 2-5 % of the whole cell proteins, with t he higher levels being recovered from non-growing cells. The two prote ins, gpi 23 and gpi 63, were purified to near homogeneity through Trit on X-114/water partitioning followed by preparative SDS/PAGE. The lipi d components of the GPI anchors were determined by chemical and enzymi c hydrolysis. Both proteins were anchored by ceramides, with the princ ipal long-chain base being C-18 sphinganine containing an O-methyl gro up at the 3 position, O-Methylation was shown not to be an artifact of hydrolysis. When T. mimbres was cultured at 15 degrees C, the ceramid e fatty acid component of the GPI anchors was principally palmitic aci d (75 % in gpi 23 and 76 % in gpi 63). GPI anchors from 28 degrees C-g rown cells contained mainly stearic acid (79 % in gpi 23 and 70 % in g pi 63). Temperature change had little effect on the long-chain-base co mposition. The direction of temperature-induced lipid change in the pr otein-bound anchors was the same as found in the inositolphosphorylcer amide putative precursors of the protein anchors described in the acco mpanying paper [Hung, Ko and Thompson (1995) Biochem. J. 307, 107-113] , but the detailed fatty acid compositions of the precursors and the p rotein-bound lipids were quite different. The precise metabolic regula tion of anchor lipid chain length supports the concept that compositio n of the lipid anchor is important in the function and/or metabolism o f the anchored protein.