Proprotein interaction with the GPI transamidase

AbstractFor characterizing how the glycosylphosphatidylinositol (GPI) transamidase complex functions, we exploited a two‐step miniPLAP (placental alkaline phosphatase) in vitro translation system. With this system, rough microsomal membranes (RM) containing either [35S]‐labeled Gaa1p or epitope‐tagged Gpi8p, alternative components of the enzymatic complex, were first prepared. In a second translation, unmodified or mutant miniPLAP mRNA was used such that [35S]‐labeled native or variant miniPLAP nascent protein was introduced. Following this, the RM were solubilized and anti‐PLAP or anti‐epitope immunoprecipitates were analyzed. With transamidase competent HeLa cell RM, anti‐PLAP or anti‐epitope antibody coprecipitated both Gaa1p and Gpi8p consistent with the assembly of the proprotein into a Gaa1p:Gpi8p‐containing complex. When RM from K562 mutant K cells which lack Gpi8p were used, anti‐PLAP antibody coprecipitated Gaa1p. The proprotein coprecipitation of Gaa1p increased with a nonpermissive GPI anchor addition (ω) site. In contrast, if a miniPLAP mutant devoid of its C‐terminal signal was used, no coprecipitation occurred. During the transamidation reaction, a transient high Mr b