Access Type

Open Access Dissertation

Date of Award

January 2018

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry

First Advisor

Tamara L. Hendrickson

Abstract

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.

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