The soluble domains of Gpi8 and Gaa1, two subunits of glycosylphosphatidylinositol transamidase (GPI-T), assemble into a complex

摘要

Glycosylphosphatidylinositol transamidase (GPI-T) catalyzes the post-translational addition of the GPI anchor to the C-terminus of some proteins. In most eukaryotes, Gpi8, the active site subunit of GPI-T, is part of a hetero-pentameric complex containing Gpi16, Gaal, Gpi17, and Gabl. Gpi8, Gaal, and Gpil6 co purify as a heterotrimer from Saccharomyces cerevisiae, suggesting that they form the core of the GPI-T. Details about the assembly and organization of these subunits have been slow to emerge. We have previously shown that the soluble domain of S. cerevisiae Gpi8 (Gpi8(23-306)) assembles as a homodimer, similar to the caspases with which it shares weak sequence homology (Meitzler, J. L. et al., 2007). Here we present the characterization of a complex between the soluble domains of Gpi8 and Gaal. The complex between GST-Gpi(823-306) (a) and Hiss-Gaa1(50-343) (beta) was characterized by native gel analysis and size exclusion chromatography (SEC) and results are most consistent with an alpha(2)beta(2) stoichiometry. These results demonstrate that Gpi8 and Gaa1 interact specifically without a requirement for other subunits, bring us closer to determining the stoichiometry of the core subunits of GPI-T, and lend further credence to the hypothesis that these three subunits assemble into a dimer of a trimer. (C) 2017 Elsevier Inc. All rights reserved.