title: Proprotein interaction with the GPI transamidase.
abstract: For 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 [(35)S]-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 [(35)S]-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 (omega) 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 band forms. To definitively characterize this product, RM from K cells transfected with FLAG-tagged GPI8 were employed. Western blots of anti-FLAG bead isolates of solubilized RM from the cells showed that the high Mr band corresponded to Gpi8p covalently bound to miniPLAP. Loss of the band following hydrazinolysis demonstrated that the two components were associated in a thioester linkage. The data indicate that recognition of the proprotein involves Gaa1p, that the interaction with the complex does not depend on a permissive omega site, and that Gpi8p forms a thioester intermediate with the proprotein. The method could be useful for rapid analysis of nascent protein interactions with transamidase components, and possibly for helping to prepare a functional in vitro transamidase system.
authors.affiliation: Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 44106, USA.
authors.forename: Rui
authors.initials: R
authors.lastname: Chen
authors.forename: Vernon
authors.initials: V
authors.lastname: Anderson
authors.forename: Yukio
authors.initials: Y
authors.lastname: Hiroi
authors.forename: M Edward
authors.initials: ME
authors.lastname: Medof
mesh_terms: D000217:Acyltransferases / Q000737:chemistry / Q000378:metabolism*; D000469:Alkaline Phosphatase / Q000737:chemistry; D000595:Amino Acid Sequence; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000378:metabolism*; D003902:Detergents; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D011233:Precipitin Tests; D011257:Pregnancy Proteins / Q000737:chemistry; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000378:metabolism*; D021382:Protein Sorting Signals; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D013462:Sulfur Radioisotopes; D014162:Transfection
chemical_list: D015815:Cell Adhesion Molecules; D003902:Detergents; C093316:GPAA1 protein, human; D008562:Membrane Glycoproteins; C491190:PIGK protein, human; D011257:Pregnancy Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D013462:Sulfur Radioisotopes; D000217:Acyltransferases; C081007:COOH-terminal signal transamidase; C101480:miniplacental alkaline phosphatase; D000469:Alkaline Phosphatase