CYTODIFFERENTIATION IN TETRAHYMENA-VORAX IS LINKED TO GLYCOSYL-PHOSPHATIDYLINOSITOL-ANCHORED PROTEIN ASSEMBLY

The role of glycosyl-PtdIns (GPI)-anchored proteins in the cyto-differ entiation of Tetrahymena vorax was examined. Labelling of cells with [ H-3]myristate or [H-3]palmitate followed by electrophoresis showed an array of proteins carrying covalently bound lipids. Electrophoresis of protein from cells labelled with the GPI-anchor components [H-3]Ins a nd [C-14]ethanolamine revealed three polypeptides on fluorograms which have apparent molecular masses of approx. 28, 50 and 82 kDa. Labelled lipid associated with these polypeptides was susceptible to release b y in vitro exposure to Bacillus thuringiensis PtdIns-specific phosphol ipase C (PI-PLC). Using labelled fatty acids, cells induced to differe ntiate showed altered GPI-anchored protein-labelling patterns in compa rison with undifferentiated control cells, with a heavily labelled 32 kDa band appearing upon differentiation. Pre-incubation of cells in 10 mM D-mannosamine, an inhibitor of GPI incorporation into protein, res ulted in a reduction of the incorporation of label into the three GPI- anchored proteins, nearly complete inhibition of differentiation and a reduction in the rate of digestive vacuole formation. A 50% inhibitio n of differentiation was obtained using 500 muM mannosamine. The inhib itory impact Of D-mannosamine on differentiation could be competitivel y and completely reversed by the inclusion of D-mannose, but not D-glu cose. Neither glucosamine nor tunicamycin inhibited differentiation. I ncubation of cells in PI-PLC (5 units/ml) plus the differentiation ind ucer resulted in an acceleration of differentiation and generally high er percentages of differentiated cells versus controls.