Studies Concerning The Function Of Gpi17p, A Subunit Of The Glycosylphosphatidylinositol: Protein Transamidase Complex In Saccharomuces Cerevisiae

Many glycoproteins of lower and higher eucaryotes are attached to the plasma membrane by means of a glycosylphosphatidylinositol(GPI) . GPI-anchored proteins are synthesized on membrane-bound ribosomes. Upon translocation of the pro-protein across the endoplasmic reticulum membrane, GPI: protein transamidase (GP1T) recognize and removes the carboxy terminal GPI signal sequence and attaches a GPI molecule to the newly exposed carboxy terminal amino acid . The GPIT-catalyzed reaction represents the final step in the assembly of a GPl-anchored protein, and provides the critical post-translational modification of this class of proteins that allows them to gain entry into ER-derived transport vesicles for delivery to the cell surface?GPI anchoring of proteins is essential for the growth of Saccharomuces cerevisiae. GPIT in yeast is a minimally heterotetrameric membrane protein complex composed of the subunits GpiSp (-45 kDa), Gaalp (-68 kDa), Gpil7p (-61kDa), and Gpil6p (-65 kDa). All these four components are all required for transamidase function. Now, except GPI8, GAA1, GPI 16 and GPI 17 share no homology with any proteins of known function and their functional role in GPIT action is unclear. In this paper, a series of genetics and biochemical experiments were proceeded to discover the function of GPI 17. The results were as follows:1. Dominant negative alleles of Gpil7p were produced by random PCR mutagenesis of three conserved fragments of Gpi17p:1) Having generated the plasmid library which contains 100000 plasmids harboring Gpi17 mutants then screened from 8000 clones, we got 25 strong and 3 very strong dominant negative alleles. And the laters were in the second andthe third conserved fragments. Sequencing result showed that we had gotten the single mutants.2) The growth curves for different cells show that overexpression of Gpil7p has no lethal effect on the yeast cells and only that of the dominant negative mutants can cause cells not grow.3) FOA (fluoroorotic acid) treatment with the dominant negative mutants showed that the dominant negative effects of mutants were caused by the presence of the gpi17 vector, not by another mutation located somewhere in the genome and in a gene involved in the natural resistance against copper.2. The dominant negative mutants were analysed by the biochemical method to detect the function of GPI17:1) The cells harboring the dominant negative mutants of Gpil7p were radio-labeled , analytic results showed that there were accumulation of free GPI lipids and no new lipid was produced. This means that GPI17 has no relation with the biosynthesis of GPI, but is involved in the GPI anchoring process.2) Measured the survival curves for the cells overexpressing mutant alleles of Gpil7p and chosed the proper Cu2+concentration in the medium for inducing.3) Western blotting of Gas1p when mutant Gpi17p overexpressed showed that there was accumulation of immature Gas1p. This means that there is a defect in GPI-anchoing.4)When mutant Gpil7p overexpressed , it blocked the translocation of Gaslp. 5)There was no translocation defect when mutant Gpi8p overexpressed.