Regulation of Paramecium primaurelia glycosylphosphatidyl-inositol biosynthesis via dolichol phosphate mannose synthesis

A set of glycosylinositol-phosphoceramides, belonging to a family of glycos ylphosphatidyl-inositols (GPIs) synthesized in a cell-free system prepared from the free-living protozoan Paramecium primaurelia has been described. T he final GPI precursor was identified and structurally characterized as: et hanolamine-phosphate-6Man alphal-2Man alpha1-6(mannosylphosphate) Man alpha 1-4glucosamine-inositol-phospho-ceramide. During our investigations on the biosynthesis of the acid-labile modification, the additional mannosyl phosp hate substitution, we observed that the use of the nucleotide triphosphate analogue GTP gammaS (guanosine 5'-O(thiotriphosphate)) blocks the biosynthe sis of the mannosylated GPI glycolipids. We show that GTP gammaS inhibits t he synthesis of dolichol-phosphate-mannose, which is the donor of the manno se residues for GPI biosynthesis. Therefore, we investigated the role of GT P binding regulatory 'G' proteins using cholera and pertussis toxins and an intracellular second messenger cAMP analogue, 8-bromo-cAMP. All the data o btained suggest the involvement of classical heterotrimeric G proteins in t he regulation of GPI-anchor biosynthesis through dolichol-phosphate-mannose synthesis via the activation of adenylyl cyclase and protein phosphorylati on. Furthermore, our data suggest that GTP gammaS interferes with synthesis of dolichol monophosphate, indicating that the dolichol kinase is regulate d by the heterotrimeric G proteins. (C) 2001 Societe francaise de biochimie et biologie moleculaire/Editions scientifiques et medicales Elsevier SAS. All rights reserved.