THE AFFECTED GENE UNDERLYING THE CLASS-K GLYCOSYLPHOSPHATIDYLINOSITOL(GPI) SURFACE PROTEIN DEFECT CODES FOR THE GPI TRANSAMIDASE

The final step in glycosylphosphatidylinositol (GPI) anchoring of cell surface proteins consists of a transamidation reaction in which preas sembled GPI donors are substituted for C-terminal signal sequences in nascent polypeptides. In previous studies we described a human K562 ce ll mutant, termed class K, that accumulates fully assembled GPI units but is unable to transfer them to N-terminally processed proproteins. In further work we showed that, unlike wild-type microsomes, microsome s from these cells are unable to support C-terminal interaction of pro proteins with the small nucleophiles hydrazine or hydroxylamine, and t hat the cells thus are defective in transamidation, In this study, usi ng a modified recombinant vaccinia transient transfection system in co njunction with a composite cDNA prepared by 5' extension of an existin g GenBank sequence, we found that the genetic element affected in thes e cells corresponds to the human homolog of yGPI8, a gene affected in a yeast mutant strain exhibiting similar accumulation of GPI donors wi thout transfer, hGPI8 gives rise to mRNAs of 1.6 and 1.9 kb, both enco ding a protein of 395 amino acids that varies in cells with their abil ity to couple GPIs to proteins, The gene spans approximate to 25 kb of DNA on chromosome 1, Reconstitution of class K cells with hGPI8 aboli shes their accumulation of GPI precursors and restores C-terminal proc essing of GPI-anchored proteins, Also, hGPI8 restores the ability of m icrosomes from the mutant cells to yield an active carbonyl in the pre sence of a proprotein which is considered to be an intermediate in cat alysis by a transamidase.