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.
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http://dx.doi.org/10.1016/j.abb.2017.09.006 | DOI Listing |
Biochemistry
July 2022
Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, Michigan 48202, United States.
Glycosylphosphatidylinositol (GPI) anchoring of proteins is a eukaryotic, post-translational modification catalyzed by GPI transamidase (GPI-T). The GPI-T is composed of five membrane-bound subunits: Gpi8, Gaa1, Gpi16, Gpi17, and Gab1. GPI-T has been recalcitrant to structure and function studies because of its complexity and membrane-solubility.
View Article and Find Full Text PDFArch Biochem Biophys
November 2017
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. Electronic address:
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.
View Article and Find Full Text PDFSci Rep
April 2017
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Glycosylphosphatidylinositol (GPI) transamidase (GPIT), the enzyme that attaches GPI anchors to proteins as they enter the lumen of the endoplasmic reticulum, is a membrane-bound hetero-pentameric complex consisting of Gpi8, Gpi16, Gaa1, Gpi17 and Gab1. Here, we expressed and purified the luminal domain of Saccharomyces cerevisiae (S. cerevisiae) Gpi8 using different expression systems, and examined its interaction with insect cell expressed luminal domain of S.
View Article and Find Full Text PDFMol Plant Microbe Interact
November 2016
1 Martin-Luther-Universität Halle-Wittenberg, Naturwissenschaftliche Fakultät III, Institut für Agrar- und Ernährungswissenschaften, Phytopathologie und Pflanzenschutz, and.
Glycosylphosphatidylinositol (GPI) anchoring of proteins is one of the most common posttranslational modifications of proteins in eukaryotic cells and is important for associating proteins with the cell surface. In fungi, GPI-anchored proteins play essential roles in cross-linking of β-glucan cell-wall polymers and cell-wall rigidity. GPI-anchor synthesis is successively performed at the cytoplasmic and the luminal face of the ER membrane and involves approximately 25 proteins.
View Article and Find Full Text PDFJ Biol Chem
February 2004
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706-1544, USA.
Glycosylphosphatidylinositol (GPI)-anchored proteins are synthesized as precursor proteins that are processed in the endoplasmic reticulum by GPI transamidase (GPIT). Human GPIT is a multisubunit membrane-bound protein complex consisting of Gaa1, Gpi8, phosphatidylinositol glycan (PIG)-S, PIG-T, and PIG-U. The enzyme recognizes a C-terminal signal sequence in the proprotein and replaces it with a preformed GPI lipid.
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