| Glycosylphosphatidylinositol (GPI) anchoring is an essential eukaryotic post-translational modification that uses a glycolipid anchor as an alternative mechanism to attach select proteins to the membrane without the aid of a peptidyl transmembrane domain. GPI-anchored proteins are localized on the surface of eukaryotic cells where they carry out many important functions. Thus GPI transamidase (GPI-T), the enzyme that attaches the GPI anchor to these proteins, is essential for normal cell function.;GPI-T is a multi-subunit membrane-bound enzyme that catalyzes the replacement of a C-terminal signal sequence in the proprotein substrate with the GPI anchor in the endoplasmic reticulum (ER). It is composed of at least three subunits: Gpi8 (PIG-K), Gaa1 (GAA1), and Gpi16 (PIG-T). (Yeast subunit names are listed first with the names of the human orthologs given in parentheses.) These subunits are conserved in all eukaryotes. Gpi8 is the catalytic subunit of the enzyme. Two additional subunits are part of the human GPI-T and are also found in yeast: Gpi17 (PIG-S) and Gab1 (PIG-U). Each of the four non-catalytic subunits is essential for anchor attachment. Genetic and biochemical analyses have led to postulated, but as yet unverified, roles for some of them.;GPI-T is a complex enzyme, yet we know next to nothing about its mode of action. A cysteine protease-like mechanism has been proposed within Gpi8, however roles for the remaining subunits remain elusive. GPI anchoring is the last major post-translational modification whose function is poorly understood. Moreover, the significance of the GPI anchor in a number of disease states from cancer to prion infectivity has recently been uncovered. Thus the importance of GPI research is not purely of academic interest but is medically important as well.;We present herein our efforts towards the expression and characterization of the soluble domain of Gaa1 (Gaa150-343), an essential GPI-T subunit in yeast and humans. The truncated soluble subunit Gpi823-306 has been shown to homodimerize and to interact with Gaa150-343 . Using electrospray ionization ion mobility separation mass spectrometry (ESI-IMS-MS), we have determined the stoichiometry of the Gpi823-306 :Gaa150-343 complex to be α2β 2. This result suggests that wild-type GPI-T assembles as a symmetrical dimer.;Results are also presented for the ongoing development of an in vitro fluorescence assay to measure GPI-T activity. Peptide substrates with varying degrees of truncation into the GPI signal sequence were tested for efficient processing by GPI-T. These results reveal an interesting pattern in permitted lengths of GPI signal sequences, which bears similarity to the substrate tolerance of oligosaccharyltransferase (OST), the enzyme responsible for N-linked glycosylation. Finally, progress towards the development of a complementary in vivo reporter assay is also presented. |
