Endoplasmic reticulum proteins involved in glycosylphosphatidylinositolâanchor attachment: Photocrosslinking studies in a cellâfree system
2001
Vidugiriene, Jolanta | Vainauskas, Saulius | Johnson, Arthur E. | Menon, Anant K.
Assembly of glycosylphosphatidylinositol (GPtdIns)âanchored proteins requires translocation of the nascent polypeptide chain across the endoplasmic reticulum (ER) membrane and replacement of the Câterminal signal sequence with a GPtdIns moiety. The anchoring reaction is carried out by an ER enzyme, GPtdIns transamidase. Genetic studies with yeast indicate that the transamidase consists of a dynamic complex of at least two subunits, Gaa1p and Gpi8p. To study the GPtdInsâanchoring reaction, we used a small reporter protein that becomes GPtdInsâanchored when the corresponding mRNA is translated in the presence of microsomes, in conjunction with siteâspecific photocrosslinking to identify ER membrane components that are proximal to the reporter during its conversion to a GPtdInsâanchored protein. We generated variants of the reporter protein such that upon in vitro translation in the presence of Neâ(5âazidoâ2ânitrobenzoyl)âlysylâtRNA, photoreactive lysine residues would be incorporated in the protein specifically near the GPtdInsâattachment site. We analyzed photoadducts resulting from UV irradiation of the samples. We show that proproteins can be crosslinked to the transamidase subunit Gpi8p, as well as to ER proteins of molecular mass ≈â60âkDa, ≈â70âkDa, and ≈â120âkDa. The identification of a photoadduct between a proprotein and Gpi8p provides the first direct evidence of an interaction between a proprotein substrate and one of the genetically identified transamidase subunits. The ≈â70âkDa protein that we identified may correspond to the other subunit Gaa1p, while the other proteins possibly represent additional, hitherto unidentified subunits of the mammalian GPtdIns transamidase complex.
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