MAMMALIAN GLYCOPHOSPHATIDYLINOSITOL ANCHOR TRANSFER TO PROTEINS AND POSTTRANSFER DEACYLATION

The glycophosphatidylinositol (GPI) anchors of proteins expressed on h uman erythrocytes and nucleated cells differ with respect to acylation of an inositol hydroxyl group, a structural feature that modulates th eir cleavability by PI-specific phospholipase C (PI-PLC). To determine how this GPI anchor modification is regulated, the precursor and prot ein-associated GPIs ins two K562 cell transfectants (ATCC and .48) exh ibiting alternatively PI-PLC-sensitive and resistant surface proteins were analyzed and the temporal relationship between GPI protein transf er and acquisition of PI-PLC sensitivity was determined, Nondenaturing PAGE analyses demonstrated that, whereas in .48 transfectants the GPI anchors in decay accelerating factor (DAF) and placental alkaline pho sphatase (PLAP) were > 95% acylated, in ATCC transfectants, they were 60 and 33% unsubstituted, respectively. In contrast, TLC analyses reve aled that putative GPI donors in the two lines mere identical and were greater than or equal to 95% acylated, Studies of de novo DAF biosynt hesis in HeLa cells bearing proteins with > 90% unacylated anchors sho wed that within 5 min at 37 degrees C (or at 18 degrees C, which does not permit endoplasmic reticilum exit), > 50% of the anchor in nascent 44-kDa proDAF protein exhibited PI-PLC sensitivity. In vitro analyses of the microsomal processing of miniPLAP, a truncated FLAP reporter p rotein, demonstrated that the anchor donor initially transferred to pr ominiPLAP was acylated and then progressively was deacylated, These fi ndings indicate that (i) the anchor moiety that initially transfers to nascent proteins is acylated, (ii) inositol acylation in mature surfa ce proteins is regulated via posttransfer deacylation, which in genera l is cell-specific but also can be protein-dependent, and (iii) deacyl ation occurs in the endoplasmic reticulum immediately after GPI transf er.