13C NMR studies of acetate metabolism during sporulation of Saccharomyces cerevisiae.

Abstract

The sporulation of Saccharomyces cerevisiae in the presence of [2-13C]acetate was studied by 13C NMR spectroscopy. The fate of 13C label was analyzed in vivo and in cell extracts. During the first 4 hr of sporulation the major metabolite produced from [2-'3C]acetate utilization was glutamate. From the labeling pattern observed it is concluded that both the tricarboxylic acid cycle and the glyoxylate cycle are operating. After about 4 hr trehalose is made. Comparison of the doublet/singlet ratios for C-1, 1' and C-6, 6' of trehalose shows a steady drop in the ratio of C-1, C-2-coupled species over trehalose labeled only at C-1 in the C-1, 2 segment of the molecule. The negative correlation of this ratio with that for the C-5, 6 segment indicates a cycling of glucose through the hexose monophosphate shunt. Subsequently fatty acid biosynthesis commences. Large amounts of saturated fatty acid were made. There were conspicuous differences observed in the metabolism of [2-'3C]acetate between sporulating and vegetatively growing cells.Diploid strains of Saccharomyces cerevisiae can undergo a developmental switch from vegetative growth to the alternative processes of meiosis and ascospore formation. This switch is dependent on the presence of two alleles of the mating-type locus MAT a and MAT a, it can only occur during a particular phase of the cell division cycle, and it is subject to regulation by the nutritional conditions operating (1). The molecular mechanisms responsible for this developmental switch are not known, and the basic changes in metabolism causing or accompanying sporulation are poorly understood and the subject of conflicting reports.The present study was done to determine more precisely the metabolic changes that accompany sporulation. For this NMR spectroscopy is ideal because it enables an extensive and detailed analysis of the basic metabolic processes that occur. Moreover, yeast sporulation is very well suited to NMR analysis: 3Clabeled acetate compounds are readily available, and sporulation occurs solely in acetate (2). From NMR studies it is possible to identify labeled intermediates, to quantitate some individual enzyme transformations, and to gain a broad insight into those pathways that are operating. EXPERIMENTAL S. cerevisiae strain S41, which has the genotype MATa HO arg 4-1 cyh 1 MATa HO arg 4-1 cyh 1 was grown at 30°C with shaking in a complex medium containing per liter: 10 g of yeast extract, 20 g of peptone, 10 g of potassium acetate (or [2-'3C]sodium acetate), 1 g of glucose, 0.2 g of adenine, and 0.2 g of uracil. Sporulation medium contained per liter: 10 g of sodium acetate, 0.2 g of adenine, 0.2 g of uracil in 100 mM potassium chloride, and 0.15 g of benzylpenicillin. Sporulation was induced by the method of Fast (2).At intervals during sporulation, 1-1. 25 X 109 cells were harvested by filtration and fixed in ice-cold 5% (wt/vol) perchloric acid. After 1 hr at 0°C the material was centrifuged for 5 min at 5,000 x g. Perchloric acid-soluble material was adjusted to pH 7...

A study of branched‐chain amino acid aminotransferase and isolation of mutations affecting the catabolism of branched‐chain amino acids in Saccharomyces cerevisiae

“…The carbon atoms derive from the acetate of the sporulation medium, but the origin of the amino groups has always been unclear. The timing of the increase in branched-chain amino acid aminotransferase matches that of the bulk of new glutamate synthesis and is mirrored by the intracellular levels of branched-chain amino acids ( [20,22] and unpublished observations), and could certainly provide some, if not all, of the amino groups required for the synthesis of glutamate.…”

“…Hence, the increase in branchedchain amino acid aminotransferase is not a sporulationspecific event but the enzyme is essential for sporulation because baa1 homozygous diploids cannot sporulate. One of the earliest metabolic changes that occurs upon initation of sporulation is the synthesis of a large amount of glutamate [20,21]. The carbon atoms derive from the acetate of the sporulation medium, but the origin of the amino groups has always been unclear.…”

A Study of the Role of the Hexose Monophosphate Pathway with Respect to Fatty Acid Biosynthesis in Sporulation of Saccharomyces cerevisiae

“…The same is t&e for C-6. Hence comparison of the doublet/singlet ratios for C-1,l' and C-6,6' resonances in tfehalose of yeast cells sporulating in [2-13C]acetate reveals the degree of cycling of gluconeogenically synthesized glucose 6-phosphate through the hexose monophosphate pathway (Dickinson et al, 1983). This can be explained as follows: [2-13C]acetate entering the cell will give rise to phosphoeno(2,3-l 3C,]pyruvate and phosphoeno(1 ,2,3-13C3]pyruvate.…”

“…There is an absolute requirement for an intact sequence of gluconeogenic enzymes to accomplish this phase of the process (Dickinson & Williams, 1986). Subsequently fatty acid synthesis begins, concomitant with onset of operation of the hexose monophosphate pathway (Dickinson et al, 1983). We reasoned that the apparent coupling of fatty acid biosynthesis with the involvement of the hexose monophosphate pathway is more than fortuitous coincidence.…”

“…Previous work aimed at characterizing the metabolism of sporulation showed that initiation of sporulation involves a rapid change in the operation of the tricarboxylic acid and glyoxylate cycles such that the intracellular concentration of glutamate increases (Dickinson et al, 1983(Dickinson et al, , 1985. At about 4 h, net glutamate synthesis ceases and trehalose synthesis is initiated via glyoxalate cycle derived intermediates.…”