Glycosylphosphatidylinositol (GPI) anchoring of proteins is a eukaryotic, post-translational modification catalyzed by GPI transamidase (GPI-T). The Saccharomyces cerevisiae GPI-T is composed of five membrane-bound subunits: Gpi8, Gaa1, Gpi16, Gpi17, and Gab1. GPI-T has been recalcitrant to in vitro structure and function studies because of its complexity and membrane-solubility. Furthermore, a reliable, quantitative, in vitro assay for this important post-translational modification has remained elusive despite its discovery more than three decades ago.Three recent reports describe the structure of GPI-T from S. cerevisiae and humans, shedding critical light on this important enzyme and offering insight into the functions of its different subunits. Here, we present the purification and characterization of a truncated soluble GPI-T heterotrimer complex (Gpi8_23-306, Gaa1_50-343, and Gpi16_20-551) without transmembrane domains. Using this simplified heterotrimer, we report the first quantitative method to measure GPI-T activity in vitro and demonstrate that this soluble, minimalistic GPI-T retains transamidase activity. These results contribute to our understanding of how this enzyme is organized and functions, and provide a method to screen potential GPI-T inhibitors.

• PMID: 35730892
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Journal Article | Research Support, Non-U.S. Gov't

Authors

Ness TJ, Gamage DG, Ekanayaka SA, Hendrickson TL

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