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Breaking the boundaries of the GPI transamidase complex (LB147)
Author(s) -
G Gamage Dilani,
Hendrickson Tamara
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.lb147
Subject(s) - protein subunit , enzyme , immunoprecipitation , transmembrane protein , chemistry , glycosylation , transmembrane domain , function (biology) , biochemistry , microbiology and biotechnology , biology , membrane , receptor , gene
Breaking the boundaries of the GPI transamidase complex Dilani G. Gamage , and Tamara L. Hendrickson Department of Chemistry, Wayne State University, Detroit, Michigan 48202 Glycosylphosphatidylinositol transamidase (GPI‐T) is a complicated, membrane‐bound, multi‐subunit enzyme that catalyzes an essential post‐translational modification. 1‐3 This enzyme attaches GPI anchors to the C‐termini of various proteins that contain a proper GPI‐T signal sequence. Gpi8, Gaa1, Gpi16, Gpi17 and Gab1 are the five known subunits that may encompass the fungal GPI‐T; Gpi8 is the catalytic subunit but the functions of the other subunits remain largely unknown. 5 , 6 In humans, different GPI‐T subunits are upregulated in different cancers, making GPI‐T a target for cancer research. 7 , 8 However, in spite of the importance of this enzyme, little is known about how it assembles into an active enzyme complex, the stoichiometry of this complex, or the roles of the different components. Here we use soluble domains of the three core subunits (Gpi8, Gpi16 and Gaa1) to investigate the stoichiometry of the enzyme as well as to study the functions of each subunit. Results are shown for the homodimerization of the soluble domain of Gpi8 and the effect of N‐linked glycosylation on Gpi8 homodimerization. Co‐immunoprecipitation of the soluble domains of Gpi8:Gaa1, Gpi8:Gpi16 and Gpi8:Gpi16:Gaa1 discloses a minimal arrangement for these subunits in the absence of their transmembrane domains. Our results will help to elucidate the function and resolve the complexity of GPI‐T. To better understand how changes in expression affect GPI‐T activity, and as a model for this enzyme in cancer, we have also developed an in vivo strategy to monitor and quantify the effect of subunit overexpression on cell surface presentation of GPI‐anchored proteins in Saccharomyces cerevisiae . Grant Funding Source : Wayne State University and the American Cancer Society.