Open Access Dissertation
Date of Award
Tamara L. Hendrickson
Glycosylphosphatidylinositol (GPI) anchoring of proteins is a eukaryotic, posttranslational
modification catalyzed by GPI transamidase (GPI-T). The Saccharomyces
cerevisiae GPI-T is composed of five membrane-bound subunits: Gaa1, Gpi8, Gpi16,
Gpi17, and Gab1. Structural and functional studies have been hindered by the
complexity of this enzyme. Conditions to purify the Gpi8:Gaa1:Gpi16 GPI-T heterotrimer
from yeast have been reported, but an understanding of the subunit functions,
interactions, and stoichiometry remain unclear. Furthermore, a reliable, quantitative, in
vitro assay for this important post-translational modification has remained elusive for
nearly three decades.
Our laboratory has developed an in vitro peptide cleavage assay that correlates
changes in fluorescence to GPI-T activity. Using this peptide cleavage assay, it was
demonstrated that the purified, full-length GPI-T retains activity, providing the first
method to measure GPI-T activity in a quantitative, time-dependent manner.
This dissertation research presents the characterization of the soluble domains of the
GPI-T heterotrimeric complex, composed of Gpi823-306, Gaa150-343, and Gpi1620-551. Each
soluble domain interacts with one another without the need for the third subunit. This soluble GPI-T heterotrimer can be purified as one complex without its transmembrane
domains. Most importantly, this simplified heterotrimer retains transamidase activity,
demonstrating that these three subunits comprise the functional core of GPI-T.
These results contribute to our understanding of how this enzyme is structurally
organized, provide a method to screen potential GPI-T inhibitors, and open the door to
further understand how GPI-T is involved in normal cellular function and pathogenesis.
Ness, Travis, "Investigation Of The Saccharomyces Cerevisiae Gpi Transamidase: Insights Into Its Activity And Subunit-Subunit Interactions" (2018). Wayne State University Dissertations. 1952.