POSSIBLE LOCALIZATION OF DOLICHOL-DEPENDENT MANNOSYLTRANSFERASE OF TRYPANOSOMA-BRUCEI TO THE ROUGH ENDOPLASMIC-RETICULUM

The glycosylphosphatidylinositol membrane anchor of variant surface gl ycoprotein of the African trypanosome Trypanosoma brucei contains seve ral mannosyl residues for which dolichol phosphoryl mannose is suppose d to be the precursor; this itself is probably synthesised by a dolich ol-dependent mannosyltransferase. We have characterised and localised a mannosyltransferase activity of T. brucei which transfers mannose fr om GDP-[C-14]mannose to exogenously added dolichyl phosphate. The enzy me was saturable for both its substrates and had a K-m of 7.8 mu M and 3.3 mu M, respectively, for dolichyl phosphate and GDP-mannose. Manno syltransferase was labile at 37 degrees C in the presence of Triton X- 100, but its activity remained constant for at least 60 min at tempera tures between 10-15 degrees C. The enzyme was inhibited by amphomycin and this inhibition was potentiated by the presence of 10 mM CaCl2. Af ter subcellular fractionation of cell homogenates by differential cent rifugation, mannosyltransferase was recovered mainly in the microsomal fraction and its distribution was very similar to that of RNA, a mark er for the rough endoplasmic reticulum. After isopycnic centrifugation in a linear sucrose gradient the distribution of mannosyltransferase also resembled that of RNA. Both constituents exhibited a shift toward s lower densities after pre-treatment of microsomal membranes with ino rganic pyrophosphate, while other membrane markers such as acid phosph atase and nucleoside diphosphatase did not. It is concluded that the f ormation of dolichol phosphoryl mannose from GDP-mannose and dolichyl phosphate in T. brucei occurs mainly in the rough endoplasmic reticulu m.