Gamage Dilani G, Varma Yug, Meitzler Jennifer L, Morissette Rachel, Ness Travis J, Hendrickson Tamara L
Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, MI 48202, USA.
Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, MI 48202, USA; Department of Chemistry, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA.
Arch Biochem Biophys. 2017 Nov 1;633:58-67. doi: 10.1016/j.abb.2017.09.006. Epub 2017 Sep 8.
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, Gaa1, Gpi17, and Gab1. Gpi8, Gaa1, and Gpi16 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) 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 Gaa1. The complex between GST-Gpi8 (α) and His-Gaa1 (β) was characterized by native gel analysis and size exclusion chromatography (SEC) and results are most consistent with an αβ 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.
糖基磷脂酰肌醇转酰胺酶（GPI-T）催化某些蛋白质的C末端在翻译后添加GPI锚定物。在大多数真核生物中，GPI-T的活性位点亚基Gpi8是包含Gpi16、Gaa1、Gpi17和Gab1的异源五聚体复合物的一部分。Gpi8、Gaa1和Gpi16作为异源三聚体从酿酒酵母中共同纯化出来，这表明它们构成了GPI-T的核心。关于这些亚基的组装和组织的细节一直没有很快出现。我们之前已经表明，酿酒酵母Gpi8的可溶性结构域（Gpi8）组装成同二聚体，类似于与其具有弱序列同源性的半胱天冬酶（Meitzler，J.L.等人，2007）。在这里，我们展示了Gpi8和Gaa1的可溶性结构域之间复合物的特征。通过天然凝胶分析和尺寸排阻色谱法（SEC）对GST-Gpi8（α）和His-Gaa1（β）之间的复合物进行了表征，结果与αβ化学计量比最为一致。这些结果表明，Gpi8和Gaa1特异性相互作用，不需要其他亚基，使我们更接近确定GPI-T核心亚基的化学计量比，并进一步支持了这三个亚基组装成三聚体二聚体的假设。
