This chapter describes the experimental isolation procedure and characterization of yeast glycosylphosphatidylinositol (GPI) anchoring mutants. Approaches involving screening for lack of cell-surface expression of a GPI-anchored reporter protein are not practicable in yeast, given the finding that abolition of anchor attachment to the yeast Gas1/Ggp1 protein—a well-characterized GPI-anchored protein—does not block transit of the protein to the cell surface and results in secretion of the protein. A new strategy to isolate GPI anchoring mutants that exploits the fact that yeast GPI-anchored proteins can be radiolabeled specifically with [3H]inositol, has been developed. A way to radiolabel yeast colonies with [3H]inositol and detect incorporation of that [3H]inositol into GPI-anchored proteins, and the technique is used to screen colonies of mutagenized yeast cells for those that failed to incorporate [3H]inositol into protein. The GPI strains for any growth phenotype that could be exploited to enrich a population of mutagenized yeast cells for further GPI anchoring-deficient strains, which could then be detected at higher frequency using the [3H]inositol- incorporation screen is examined in the chapter.
ASJC Scopus subject areas
- Molecular Biology