Abstract
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.
Original language | English (US) |
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Pages (from-to) | 560-571 |
Number of pages | 12 |
Journal | Methods in enzymology |
Volume | 250 |
Issue number | C |
DOIs | |
State | Published - 1995 |
Externally published | Yes |
ASJC Scopus subject areas
- Biochemistry
- Molecular Biology