S-adenosyl methionine requiring mutants in Saccharomyces cerevisiae: evidences for the existence of two methionine adenosyl transferases.

Summary Mutants requiring S-adenosyl methionine (SAM) for growth have been selected in Saccharomyces cerevisiae. Two classes of mutants have been found. One class corresponds to the simultaneous occurrence of mutations at two unlinked loci SAM1 and SAM2 and presents a strict SAM requirement for growth on any medium. The second class corresponds to special single mutations in the gene SAM2 which lead to a residual growth on minimal medium but to normal growth on SAM supplemented medium or on a complex medium like YPGA not containing any SAM. These genetic data can be taken as an indication that Saccharomyces cerevisiae possesses two isoenzymatic methionine adenosyl transferases (MAT). In addition, SAM1 and SAM2 loci have been identified respectively with the ETH-10 and ETH2 loci previously described.Biochemical evidences corroborate the genetic results. Two MAT activities can be dissociated in a wild type extract (MATI and MATII) by DEAE cellulose chromatography. Mutations at the SAM1 locus lead to the absence or to the modification of MATII whereas mutations at the SAM2 locus lead to the absence or to the modification of MATI. Moreover, some of our results seem to show that MATI and MATII are associated in vivo.     

N-terminal methionine removal and methionine metabolism in Saccharomyces cerevisiae

Methionine aminopeptidase (MetAP) catalyzes removal of the initiator methionine from nascent polypeptides. In eukaryotes, there are two forms of MetAP, type 1 and type 2, whose combined activities are essential, but whose relative intracellular roles are unclear. Methionine metabolism is an important aspect of cellular physiology, involved in oxidative stress, methylation, and cell cycle. Due to the potential of MetAP activity to provide a methionine salvage pathway, we evaluated the relationship between methionine metabolism and MetAP activity in Saccharomyces cerevisiae. We provide the first demonstration that yeast MetAP1 plays a significant role in methionine metabolism, namely, preventing premature activation of MET genes through MetAP function in methionine salvage. Interestingly, in cells lacking MetAP1, excess methionine dramatically inhibits cell growth. Growth inhibition is independent of the ability of methionine to repress MET genes and does not result from inhibition of synthesis of another metabolite, rather it results from product inhibition of MetAP2. Inhibition by methionine is selective for MetAP2 over MetAP1. These results provide an explanation for the previously observed dominance of MetAP1 in terms of N-terminal processing and cell growth in yeast. Additionally, differential regulation of the two isoforms may be indicative of different intracellular roles for the two enzymes.     

Regulation of phosphatidylinositol:ceramide phosphoinositol transferase in Saccharomyces cerevisiae.

Maximal phosphatidylinositol:ceramide phosphoinositol transferase activity was measured in yeast cells harvested during the exponential phase of growth. The addition of inositol to the growth medium resulted in a twofold increase in IPC synthase activity in cells grown in the presence or absence of exogenous choline. Enzyme activity was not regulated in yeast inositol biosynthesis regulatory mutants by the addition of inositol to the growth medium.     

Utilization by Saccharomyces cerevisiae of 5'-methylthioadenosine as a source of both purine and methionine.

Cells of the yeast Saccharomyces cerevisiae are normally impermeable to the purine nucleosides adenosine and 5'-deoxy-5'-methylthioadenosine (MTA), a product of polyamine biosynthesis. cordycepin-sensitive, adenosine-utilizing strains of S. cerevisiae were able to use MTA to fulfill an auxotrophic requirement for purine. Cordycepin-sensitive strains carrying a met5 mutation were also able to use MTA as a source of methionine. These MTA-utilizing strains of S. cerevisiae should be useful for metabolic studies of the fate of MTA.     

Biochemical and regulatory effects of methionine analogues in Saccharomyces cerevisiae.

The effect of three methionine analogues, ethionine, selenomethionine, and trifluoromethionine, on the biosynthesis of methionine in Saccharomyces cerevisiae has been investigated. We have found the following to be true. (i) A sharp decrease in the endogenous methionine concentration occurs after the addition of any one of these analogues to growing cells. (ii) All of them can be transferred to methionine transfer ribonucleic acid in vitro as well as in vivo with, as a consequence, their incorporation into proteins. In the absence of radioactive trifluoromethionine, this conclusion results from experiments of an indirect nature and must be taken as an indication rather than a direct demonstration. (iii) Ethionine and selenomethionine can be activated as homologues of S-adenosylmethionine, whereas trifluoromethionine cannot. (iv) All of them can act as repressors of the methionine biosynthetic pathway. This has been shown by measuring the de novo rate of synthesis of methionine in a culture grown in the presence of any one of the three analogues.     

Studies on the regulation of enolases and compartmentation of cytosolic enzymes in Saccharomyces cerevisiae

Three enolase isoenzymes can be distinguished after electrophoresis of yeast crude extracts. After adding glucose to derepressed cells, there was a coordinated increase in the activity of enolase I and decrease in enolase II activity. Enolase I was found to be repressed and enolase II simultaneously induced by glucose. The third enolase activity remained unchanged and was identified as that of a hybrid enzyme. Enolase catalyses the first common step of glycolysis and gluconeogenesis. Gluconeogenic enolase I shows substrate inhibition for 2-phosphoglycerate (glycolytic substrate) and glycolytic enolase II is substrate-inhibited by phosphoenolpyruvate (gluconeogenic substrate). The gluconeogenic reaction was inhibited up to 45% by physiological concentrations of fructose 1,6-bisphosphate. To test for cytological compartmentation, a method was developed for isolating microsomes. Effective enrichment of rough and smooth endoplasmic reticulum was demonstrated by electron microscopy. No evidence was obtained for any compartmentation of either enolases or other glycolytic enzymes.     

Methionine-and S-adenosyl methionine-mediated repression in a methionyl-transfer ribonucleic-acid synthetase mutant of Saccharomyces cerevisiae.

A Saccharomyces cerevisiae mutant strain unable to grow at 38 C and bearing a modified methionyl-transfer ribonucleic acid (tRNA) synthetase has been studied. It has been shown that, in this mutant, the percentage of tRNAmet charged in vivo paralleled the degree of repressibility of methionine biosynthetic enzymes by exogenous methionine. On the contrary, the repression mediated by exogenous S-adenosylmethionine does not correlate with complete acylation of tRNAmet. Althought McLaughlin and Hartwell reported previously that the thermosensitivity and the defect in the methionyl-tRNA synthetase were due to the same genetic lesion (1969), no diffenence could be found in the methionyl-tRNA synthetase activity or in the pattern of repressibility of methionine biosynthetic pathway after growth at the premissive and at a semipermissive temperature. It appears that the mutant also exhibits some other modified characters that render unlikely the existence of only one genetic lesion in this strain. A genetic study of this mutant was undertaken which led to the conclusion that the thermosensitivity and the other defects are not related to the methionyl-tRNA synthetase modification. It was shown that the modified repressibility of methionine biosynthetic enzymes by methionine and the lack of acylation of tRNAmet in vivo follow the methionyl-tRNA synthetase modification. These results are in favor of the idea that methionyl-tRNAmet, more likely than methionine, is implicated in the regulation of the biosynthesis of methionine.     

Arginine metabolism in Saccharomyces cerevisiae: subcellular localization of the enzymes.

Subcellular localization of enzymes of arginine metabolism in Saccharomyces cerevisiae was studied by partial fractionation and stepwise homogenization of spheroplast lysates. These enzymes could clearly be divided into two groups. The first group comprised the five enzymes of the acetylated compound cycle, i.e., acetylglutamate synthase, acetylglutamate kinase, acetylglutamyl-phosphate reductase, acetylornithine aminotransferase, and acetylornithine-glutamate acetyltransferase. These enzymes were exclusively particulate. Comparison with citrate synthase and cytochrome oxidase, and results from isopycnic gradient analysis, suggested that these enzymes were associated with the mitochondria. By contrast, enzymatic activities going from ornithine to arginine, i.e., arginine pathway-specific carbamoylphosphate synthetase, ornithine carbamoyltransferase, argininosuccinate synthetase, and argininosuccinate lyase, and the two first catabolic enzymes, arginase and ornithine aminotransferase, were in the "soluble" fraction of the cell.     

Misacylation of tRNA with methionine in Saccharomyces cerevisiae

Accurate transfer RNA (tRNA) aminoacylation by aminoacyl-tRNA synthetases controls translational fidelity. Although tRNA synthetases are generally highly accurate, recent results show that the methionyl-tRNA synthetase (MetRS) is an exception. MetRS readily misacylates non-methionyl tRNAs at frequencies of up to 10% in mammalian cells; such mismethionylation may serve a beneficial role for cells to protect their own proteins against oxidative damage. The Escherichia coli MetRS mismethionylates two E. coli tRNA species in vitro, and these two tRNAs contain identity elements for mismethionylation. Here we investigate tRNA mismethionylation in Saccharomyces cerevisiae. tRNA mismethionylation occurs at a similar extent in vivo as in mammalian cells. Both cognate and mismethionylated tRNAs have similar turnover kinetics upon cycloheximide treatment. We identify specific arginine/lysine to methionine-substituted peptides in proteomic mass spectrometry, indicating that mismethionylated tRNAs are used in translation. The yeast MetRS is part of a complex containing the anchoring protein Arc1p and the glutamyl-tRNA synthetase (GluRS). The recombinant Arc1p–MetRS–GluRS complex binds and mismethionylates many tRNA species in vitro. Our results indicate that the yeast MetRS is responsible for extensive misacylation of non-methionyl tRNAs, and mismethionylation also occurs in this evolutionary branch.     

The CIT3 gene of Saccharomyces cerevisiae encodes a second mitochondrial isoform of citrate synthase

We have identified a third citrate synthase gene in Saccharomyces cerevisiae which we have called CIT3 Complementation of a citrate synthase-deficient strain of Escherichia coli by lacZ  :: CIT3 gene fusions demonstrated that the CIT3 gene encodes an active citrate synthase. The CIT3 gene seems to be regulated in the same way as CIT1 , which encodes the mitochondrial isoform of citrate synthase. Deletion of the CIT3 gene in a Δ cit1 background severely reduced growth on the respiratory substrate glycerol, whilst multiple copies of the CIT3 gene in a Δ cit1 background significantly improved growth on acetate. In vitro import experiments showed that cit3p is transported into the mitochondria. Taken together, these data show that the CIT3 gene encodes a second mitochondrial isoform of citrate synthase.     

The PEP4 gene encodes an aspartyl protease implicated in the posttranslational regulation of Saccharomyces cerevisiae vacuolar hydrolases.

pep4 mutants of Saccharomyces cerevisiae accumulate inactive precursors of vacuolar hydrolases. The PEP4 gene was isolated from a genomic DNA library by complementation of the pep4-3 mutation. Deletion analysis localized the complementing activity to a 1.5-kilobase pair EcoRI-XhoI restriction enzyme fragment. This fragment was used to identify an 1,800-nucleotide mRNA capable of directing the synthesis of a 44,000-dalton polypeptide. Southern blot analysis of yeast genomic DNA showed that the PEP4 gene is unique; however, several related sequences exist in yeasts. Tetrad analysis and mitotic recombination experiments localized the PEP4 gene proximal to GAL4 on chromosome XVI. Analysis of the DNA sequence indicated that PEP4 encodes a polypeptide with extensive homology to the aspartyl protease family. A comparison of the PEP4 predicted amino acid sequence with the yeast protease A protein sequence revealed that the two genes are, in fact, identical (see also Ammerer et al., Mol. Cell. Biol. 6:2490-2499, 1986). Based on our observations, we propose a model whereby inactive precursor molecules produced from the PEP4 gene self-activate within the yeast vacuole and subsequently activate other vacuolar hydrolases.     

Physiological role of glucose-phosphorylating enzymes in Saccharomyces cerevisiae.

Starting with a mutant of Saccharomyces cerevisiae lacking glucokinase and both the hexokinase isozymes P1 and P2, strains were constructed, by genetic crosses, that carry single glucose-phosphorylating enzymes. The P1 and P2 isozymes and a structurally altered form of P1 hexokinase were partially purified from these strains. Hexokinases P1, P2, and the altered P1 enzyme, respectively, phosphorylate fructose nearly four, two, and ten times as fast as they phosphorylate glucose. Strains bearing P1 show a pronounced Pasteur reaction and phosphorylate glucose, fructose, and mannose faster than those bearing the P2 isozyme. However, there is no appreciable difference between these two hexokinases in regard to the rate and the extent of growth that they sustain. The ability of yeast to grow on a particular sugar is contingent only upon the presence of an enzyme that phosphorylates it. Glucokinase seems to be responsible for catalyzing nearly half of the glucose flux in the wild type yeast. Strains bearing glucokinase alone do show a Pasteur effect.     

SRA5 encodes the low-Km cyclic AMP phosphodiesterase of Saccharomyces cerevisiae.

sra5 mutations in Saccharomyces cerevisiae were previously shown to suppress the inefficient growth of ras2 strains on nonfermentable carbon sources and to result in deficient low-Km cyclic AMP (cAMP) phosphodiesterase activity. We have cloned SRA5 by complementation. It maps to the right arm of chromosome XV, tightly linked to PRT1, and its sequence matches the sequence of PDE2, encoding the low-Km cAMP phosphodiesterase. Disruptions of SRA5 allowed ras1 ras2 strains to grow either on rich media supplemented with cAMP or on minimal media without exogenous cAMP. sra5 strains failed to survive prolonged nitrogen starvation in the presence of exogenous cAMP.     

Polyamine uptake by DUR3 and SAM3 in Saccharomyces cerevisiae

It has been reported that GAP1 and AGP2 catalyze the uptake of polyamines together with amino acids in Saccharomyces cerevisiae. We have looked for polyamine-preferential uptake proteins in S. cerevisiae. DUR3 catalyzed the uptake of polyamines together with urea, and SAM3 was found to catalyze the uptake of polyamines together with S-adenosylmethionine, glutamic acid, and lysine. Polyamine uptake was greatly decreased in both DUR3- and SAM3-deficient cells. The K(m) values for putrescine and spermidine of DUR3 were 479 and 21.2 mum, respectively, and those of SAM3 were 433 and 20.7 mum, respectively. Polyamine stimulation of cell growth of a polyamine requiring mutant, which is deficient in ornithine decarboxylase, was not influenced by the disruption of GAP1 and AGP2, but it was diminished by the disruption of DUR3 and SAM3. Furthermore, the polyamine stimulation of cell growth of a polyamine-requiring mutant was completely inhibited by the disruption of both DUR3 and SAM3. The results indicate that DUR3 and SAM3 are major polyamine uptake proteins in yeast. We previously reported that polyamine transport protein kinase 2 regulates polyamine transport. It was found that DUR3 (but not SAM3) was activated by phosphorylation of Thr(250), Ser(251), and Thr(684) by polyamine transport protein kinase 2.     

Genetic and regulatory aspects of methionine biosynthesis in Saccharomyces cerevisiae

Methionine biosynthesis and regulation of four enzymatic steps involved in this pathway were studied in Saccharomyces cerevisiae, in relation to genes concerned with resistance to ethionine (eth(1) and eth(2)). Data presented in this paper and others favor a scheme which excludes cystathionine as an obligatory intermediate. Kinetic data are presented for homocysteine synthetase [K(m)(O-acetyl-l-homoserine) = 7 x 10(-3)m; K(i) (l-methionine) = 1.9 x 10(-3)m]. Enzymes catalyzing steps 3, 4, 5, and 9 were repressible by methionine. Enzyme 4 (homoserine-O-transacetylase) and enzyme 9 (homocysteine synthetase) were simultaneously derepressed in strains carrying the mutant allele eth(2) (r). Studies on diploid strains confirmed the dominance of the eth(2) (s) allele over eth(2) (r). Regulation of enzyme 3 (homoserine dehydrogenase) and enzyme 5 (adenosine triphosphate sulfurylase) is not modified by the allele eth(2) (r). The other gene eth(1) did not appear to participate in regulation of these four steps. Gene enzyme relationship was determined for three of the four steps studied (steps 3, 4, and 9). The structural genes concerned with the steps which are under the control of eth(2) (met(8): enzyme 9 and met(a): enzyme 4) segregate independently, and are unlinked to eth(2). These results are compatible with the idea that the gene eth(2) is responsible for the synthesis of a pleiotropic methionine repressor and suggest the existence of at least two different methionine repressors in S. cerevisiae. Implications of these findings in general regulatory mechanisms have been discussed.