SGER: Development of a Soluble Assay for the Yeast GPI Transamidase

Johns Hopkins University, Baltimore MD

Investigators

Abstract

In eukaryotes, approximately 0.5% of all encoded proteins are (or are predicted to be) modified at their C-termini with a glycosylphosphatidylinositol (GPI) anchor. Attachment of this extended glycolipid occurs in the endoplasmic reticulum, and modified proteins are ultimately tethered to the outer plasma membrane via the lipid portion of the GPI anchor. GPI anchor attachment is governed by the GPI transamidase (GPI-T), a pentameric, membrane-bound enzyme. Extensive work, in vivo, has led to a qualitative picture of the protein substrate requirements for GPI-T. At present, however, there is still no convenient, high-throughput assay for GPI-T, and thus the ability to quantify and manipulate interactions between GPI-T and its substrates in vitro remains all but inaccessible. To this end, a fluorescence assay for GPI-T will be developed using a small synthetic peptide as a substrate for GPI-T and replacing the GPI anchor with hydrazine. The strategy for assay development is based on the fact that GPI-T catalyzes a proteolysis reaction prior to anchor attachment. This assay will represent the first quantifiable in vitro assay for GPI-T and will consequently lay the foundation for future in vitro efforts to explore the GPI-T enzyme complex and the chemical complexity of this modification in detail. Development of this assay will open up diverse avenues of research in vitro into GPI anchor formation. Broader Impact The broader impact of this proposal is two-fold. First, the results will be disseminated to the greater scientific community (via publications and meetings by the PI and graduate students) so that other labs interested in GPI-T will be able to incorporate the new GPI-T assay into their own research programs. Second, students working on this project will receive a multi-disciplinary training, spanning the fields of organic chemistry, spectroscopy, enzyme kinetics and molecular biology.