The effect of the erg26-1 mutation on the regulation of lipid metabolism in Saccharomyces cerevisiae

A temperature-sensitive Saccharomyces cerevisiae mutant harboring a lesion in the ERG26 gene has been isolated. ERG26 encodes 4 alpha -carboxysterol-C 3 dehydrogenase, one of three enzymatic activities required for the convers ion of 4,4-dimethylzymosterol to zymosterol. Gas chromatography/mass spectr ometry analyses of sterols in this mutant, designated erg26-1, revealed the aberrant accumulation of a 4-methyl-4-carboxy zymosterol intermediate, as well as a novel 4-carboxysterol. Neutral lipid radiolabeling studies showed that erg26-1 cells also harbored defects in the rate of biosynthesis and s teady-state levels of mono-, di-, and triglycerides. Phospholipid radiolabe ling studies showed defects in the rate of biosynthesis of both phosphatidi c acid and phosphatidylinositol. Biochemical studies revealed that microsom es isolated from erg26-1 cells contained greatly reduced 4 alpha -carboxyst erol-C3 dehydrogenase activity when compared with microsomes from wild type cells. Previous studies have shown that loss of function mutations in eith er of the fatty acid elongase genes SUR4/EL03 or FEN1/GNS1/ELO2 can "by-pas s" the essentiality of certain ERG genes (Ladeveze, V., Marcireau, C., Delo urme, D., and Karat, F. (1993) Lipids 28, 907-912; Silve, S., Leplatois, P. , Josse, A., Dupuy, P. H., Lanau, C., Kaghad, M., Dhers, C., Picard, C., Ra hier, A, Taton, M., Le Fur, G., Caput, D., Ferrara, P., and Loison, G. (199 6) Mol Cell. Biol. 16, 2719-2727). Studies presented here have shown that t his sphingolipid-dependent "bypass" mechanism did not suppress the essentia l requirement for zymosterol biosynthesis. However, studies aimed at unders tanding the underlying physiology behind the temperature-sensitive growth d efect of erg26-1 cells showed that the addition of several antifungal compo unds to the growth media of erg26-1 cells could suppress the temperature-se nsitive growth defect. Fluorescence microscopic analysis showed that GFP-Er g26p and GFP-Erg27p fusion proteins were localized to the endoplasmic retic ulum. Two-hybrid analysis indicated that Erg25p, Erg26p, and Erg27p, which are required for the biosynthesis of zymosterol, form a complex within the cell.