A multinuclear magnetic resonance study of a cls11 mutant showing the Pet− phenotype of Saccharomyces cerevisiae

Abstract

Energetic and intermediary metabolism was studied in a Pet- mutant of Saccharomyces cerevisiae with a calcium-sensitive phenotype that shows an inability to grow when cultured in a medium containing non-fermentable substrates. The perchloric acid extracts were prepared from suspensions of cls11 mutant and wild-type cells incubated with [1,3-13C]glycerol or [2-13]acetate, and analyzed by 31P, 13C and 1H NMR. 31P- and 1H-NMR spectra showed significant differences between cls11 and wild-type cells at the level of amino acids, the storage carbohydrate trehalose (higher in mutant cells), and sugar phosphates (higher in wild-type cells). 13C-NMR spectra revealed major differences in the steady-state labelling of glutamate carbons. For incubations with [1,3-13C]glycerol, we estimated from the relative 13C enrichment of glutamate carbons that acetyl-CoA C2 is 43% C13 labelled in wild-type and 10% 13C labelled in mutant cells, respectively. For incubations with [2-13C]acetate, we calculated that the ratio of the relative flux through the glyoxylate shunt versus oxidative reactions is 58% in wild-type cells and 44% in the cls11 mutant cells. Again, a dilution of the relative enrichment of C2 of acetyl-CoA was observed in the mutant cells (89%) compared to the wild-type cells (97%). These results are discussed in terms of pleiotropic defects in non-fermentable carbon metabolism in mutant cells.