Genetic analyses involving interactions between the ergosterol biosynthetic enzymes, lanosterol synthase (Erg7p) and 3-ketoreductase (Erg27p), in the yeast Saccharomyces cerevisiae.

Protein-protein interaction studies in the Saccharomyces cerevisiae ergosterol biosynthetic pathway suggest that enzymes in this pathway may act as an integrated multienzyme complex. The yeast sterol 3-ketoreductase (Erg27p) required for C-4 demethylation of sterols has previously been shown to also be required for the function of the upstream oxidosqualene cyclase/lanosterol synthase (Erg7p); thus, erg27 mutants accumulate oxidosqualenes as precursors rather than 3-ketosterones. In the present study, we have created various mutations in the ERG27 gene. These mutations include 5 C-terminal truncations, 6 internal deletions, and 32 point mutants of which 14 were obtained by site-directed mutagenesis and 18 by random mutagenesis. We have characterized these ERG27 mutations by determining the following: Erg27 and Erg7 enzyme activities, presence of Erg27p as determined by western immunoblots, ability to grow on various sterol substrates and GC sterol profiles. Mutations of the predicted catalytic residues, Y202F and K206A, resulted in the endogenous accumulation of 3-ketosterones rather than oxidosqualenes suggesting retention of Erg7 enzyme activity. This novel phenotype demonstrated that the catalytic function of Erg27p can be separated from its Erg7p chaperone ability. Other erg27 mutations resulted in proteins that were present, as determined by western immunoblotting, but unable to interact with the Erg7 protein. We also classify Erg27p as belonging to the SDR (short-chain dehydrogenase/reductase) family of enzymes and demonstrate the possibility of homo- or heterodimerization of the protein. This study provides new insights into the role of Erg27p in sterol biosynthesis.

Lipids of brush border membrane vesicles (BBMV) from Plutella xylostella resistant and susceptible to Cry1Ac delta-endotoxin of Bacillus thuringiensis.

Plutella xylostella (PX) that were 130000-fold more resistant to Cry1Ac were selected from the susceptible strain and maintained in the laboratory. The LC50 of the susceptible strain (PXS) was 0.38 microg toxin/g diet, whereas that of the resistant strain (PXR) was 4800 microg toxin/g diet. Brush border membrane vesicles (BBMV) were prepared from both PXS and PXR. In ligand blot analysis, Cry1Ac bound to a 120-kDa protein of BBMV; however, the intensity of the band was almost equal in both strains of insect. Hence, we analyzed the lipid components of BBMV from PXS and PXR. BBMV lipids were fractionated into non-polar lipid, phospholipid, neutral glycolipid and acidic glycolipid. Neutral glycolipid content was substantially lower in the BBMV of PXR than of PXS. The same trend was observed when lipids were extracted from whole midgut instead of BBMV. Thin layer chromatography of midgut neutral glycolipids revealed the presence of more than seven components. Among the midgut neutral glycolipids, a possible hexasaccharylceramide and a possible trisaccharylceramide of PXR were less than half the level found in PXS. The other lipid fractions in PXR and PXS were similar to each other.