A DEFECT IN GLYCOSYLPHOSPHATIDYLINOSITOL (GPI) TRANSAMIDASE ACTIVITY IN MUTANT K CELLS IS RESPONSIBLE FOR THEIR INABILITY TO DISPLAY GPI SURFACE-PROTEINS

The final step in the pathway that provides for glycosylphosphatidylin ositol (GPI) anchoring of cell-surface proteins occurs in the lumen of the endoplasmic reticulum and consists of a transamidation reaction i n which fully assembled GPI anchor donors are substituted for specific COOH-terminal signal peptide sequences contained in nascent polypepti des. In previous studies we described a human K562 cell mutant line, d esignated class K, which assembles all the known intermediates of the GPI pathway but fails to display GPI-anchored proteins on its surface membrane, In the present study, we used mRNA encoding miniPLAP, a trun cated form of placental alkaline phosphatase (FLAP), in irt vitro assa ys with rough microsomal membranes (RM) of mutant K cells to further c haracterize the biosynthetic defect in this line. We found that RM fro m mutant K cells supported NH2-terminal processing of the nascent tran slational product, preprominiPLAP, but failed to show any detectable C OOH-terminal processing of the resulting prominiPLAP to GPI-anchored m iniPLAP, Proteinase K protection assays verified that NH2-terminal-pro cessed prominiPLAP was appropriately translocated into the endoplasmic reticulum lumen, The addition of hydrazine or hydroxylamine, which ca n substitute for GPI donors, to RM from wild-type or mutant cells defe ctive in various intermediate biosynthetic steps in the GPI pathway pr oduced large amounts of the hydrazide or hydroxamate of miniPLAP, In c ontrast, the addition of these nucleophiles to RM of class K cells yie lded neither of these products, These data, taken together, lead us to conclude that mutant K cells are defective in part of the GPI transam idase machinery.