Phosphatidylinositol-glycan (PI-G)-anchored membrane proteins: requirement of ATP and GTP for translation-independent COOH-terminal processing.

Placental alkaline phosphatase (PLAP) belongs to a class of proteins that are anchored to the plasma membrane by a COOH-terminal phosphatidylinositol-glycan (PI-G) moiety. Nascent forms of such proteins undergo NH2- and COOH-terminal processing to yield the mature PI-G-tailed proteins. We previously introduced a shortened engineered form of preproPLAP (preprominiPLAP) that permits monitoring in cell-free preparations its sequential processing to the pro form and then to the mature PI-G-tailed form. Previous studies were carried out by synthesizing the preproprotein cotranslationally in the presence of rough microsomal membranes (RM). Because of the complexity of the cotranslational system it was not possible to determine whether cofactors were required for processing. We have now prepared RM that are preloaded with prominiPLAP but contain little mature PI-G-tailed miniPLAP. Maximal processing requires supplementation with both ATP and GTP. Inhibitors of PI-G biosynthesis do not affect processing. Since cleavage and PI-G addition are presumably catalyzed by a transamidase, the nucleoside triphosphate requirements suggest that there are additional steps in prominiPLAP processing prior to transamidation with PI-G. These may involve translocation of the pro protein in a proper conformational state to the transamidase site.