Preparation of microsomes from yeast and GPI-GnT assay

Primary cultures of yeast strains were grown for 16 h in 4% (w/v) galactose. It was inoculated into 250 ml SD medium containing either 4% galactose or 2% glucose at 30 °C till OD_600nm of 2.0 was reached. The cells were harvested and microsomes prepared from it as described previously³⁷.

GPI-GnT activity of the microsomes was assayed as described previously³⁷. The assay produces both [6³H]GlcNAc-PI (N-acetyl glucosaminylphosphatidylinositol) and [6³H]GlcN-PI (glucosaminylphosphatidylinositol, the product of the next step of the pathway) and were detected using Bioscan AR2000 TLC scanner. The sum of the radioactive counts detected for the two products in the case of the control strain was considered to be 100% and GPI-GnT activity of all strains was calculated relative to it. In all cases averages of data from independent experiments done twice in duplicates along with standard deviations are plotted.