Tetrahymena species contain relatively prominent glycosylphosphatidylinositol (GPI)-anchored proteins as well as their putative precursor phosphatidylinositol (PI) glycans. We have characterized the lipid components of the two principal T. mimbres PI glycans. Following their purification by preparative TLC, the PI glycans were hydrolysed in methanolic HCl or NaOH, and resulting lipids were analysed by chromatography and mass spectrometry. The two PI glycans contained nearly identical lipid moieties having long-chain bases with N-linked fatty acids. The predominant long-chain base, 3-O-methylsphinganine, was first assumed to be O-methylated as an artifact of hydrolysis, but subsequently, on the basis of control experiments, it was shown to be naturally occurring. PI glycans from cells grown at 28 degrees C contained primarily palmitic acid (79%) and some stearic acid (11%), whereas the principal PI glycan from 38 degrees C-grown T. mimbres contained 65% stearic acid. In 15 degrees C-grown cells stearic acid accounted for only 2% of ceramide-bound fatty acids and was almost totally replaced by palmitic acid (95%). The distributions of fatty acids bound to T. mimbres GPI-anchored proteins [Ko, Hung and Thompson (1995) Biochem. J. 307, 115-121] were similar but not identical to those of the PI glycans described here. Temperature-induced specification of the lipid components of mature T. mimbres GPI-anchored proteins appears to be established both at the level of PI-glycan synthesis and the level of PI-glycan utilization for protein attachment.

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