We sought to create a comprehensive catalog of yeast genes whose transcript levels vary periodically within the cell cycle. To this end, we used DNA microarrays and samples from yeast cultures sync...

Comprehensive Identification of Cell Cycle–regulated Genes of the Yeast Saccharomyces cerevisiae by Microarray Hybridization

Multiplexed Protein Quantitation in Saccharomyces cerevisiae Using Amine-reactive Isobaric Tagging Reagents

Technologies to measure whole-genome mRNA abundances1,2,3 and methods to organize and display such data4,5,6,7,8,9,10 are emerging as valuable tools for systems-level exploration of transcriptional regulatory networks. For instance, it has been shown that mRNA data from 118 genes, measured at several time points in the developing hindbrain of mice, can be hierarchically clustered into various patterns (or 'waves') whose members tend to participate in common processes5. We have previously shown that hierarchical clustering can group together genes whose cis-regulatory elements are bound by the same proteins in vivo6. Hierarchical clustering has also been used to organize genes into hierarchical dendograms on the basis of their expression across multiple growth conditions7. The application of Fourier analysis to synchronized yeast mRNA expression data has identified cell-cycle periodic genes, many of which have expected cis-regulatory elements8. Here we apply a systematic set of statistical algorithms, based on whole-genome mRNA data, partitional clustering and motif discovery, to identify transcriptional regulatory sub-networks in yeast—without any a priori knowledge of their structure or any assumptions about their dynamics. This approach uncovered new regulons (sets of co-regulated genes) and their putative cis-regulatory elements. We used statistical characterization of known regulons and motifs to derive criteria by which we infer the biological significance of newly discovered regulons and motifs. Our approach holds promise for the rapid elucidation of genetic network architecture in sequenced organisms in which little biology is known.

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Systematic determination of genetic network architecture

Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway

SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box.

Sulfur amino acid biosynthesis in Saccharomyces cerevisiae involves a large number of enzymes required for the de novo biosynthesis of methionine and cysteine and the recycling of organic sulfur metabolites. This review summarizes the details of these processes and analyzes the molecular data which have been acquired in this metabolic area. Sulfur biochemistry appears not to be unique through terrestrial life, and S. cerevisiae is one of the species of sulfate-assimilatory organisms possessing a larger set of enzymes for sulfur metabolism. The review also deals with several enzyme deficiencies that lead to a nutritional requirement for organic sulfur, although they do not correspond to defects within the biosynthetic pathway. In S. cerevisiae, the sulfur amino acid biosynthetic pathway is tightly controlled: in response to an increase in the amount of intracellular S-adenosylmethionine (AdoMet), transcription of the coregulated genes is turned off. The second part of the review is devoted to the molecular mechanisms underlying this regulation. The coordinated response to AdoMet requires two cis-acting promoter elements. One centers on the sequence TCACGTG, which also constitutes a component of all S. cerevisiae centromeres. Situated upstream of the sulfur genes, this element is the binding site of a transcription activation complex consisting of a basic helix-loop-helix factor, Cbf1p, and two basic leucine zipper factors, Met4p and Met28p. Molecular studies have unraveled the specific functions for each subunit of the Cbf1p-Met4p-Met28p complex as well as the modalities of its assembly on the DNA. The Cbf1p-Met4p-Met28p complex contains only one transcription activation module, the Met4p subunit. Detailed mutational analysis of Met4p has elucidated its functional organization. In addition to its activation and bZIP domains, Met4p contains two regulatory domains, called the inhibitory region and the auxiliary domain. When the level of intracellular AdoMet increases, the transcription activation function of Met4 is prevented by Met30p, which binds to the Met4 inhibitory region. In addition to the Cbf1p-Met4p-Met28p complex, transcriptional regulation involves two zinc finger-containing proteins, Met31p and Met32p. The AdoMet-mediated control of the sulfur amino acid pathway illustrates the molecular strategies used by eucaryotic cells to couple gene expression to metabolic changes.

Metabolism of sulfur amino acids in Saccharomyces cerevisiae.

Strains resistant to the toxic analogues of sulfate, selenate and chromate have been isolated. Their genetic analysis allowed us to identify four genes. One, called MET28, encodes a transcriptional factor. The three other genes, called SUL1, SUL2 and SUL3, encode proteins involved in sulfate transport. The sequence of Sul1p and Sul2p indicate that they are integral membrane proteins exhibiting, respectively, 11 and 10 transmembrane domains. Moreover, Sul1p and Sul2p share a high degree of similarity. Sulfate transport kinetic studies made with parental and mutant strains show that, as expected from genetic results, Saccharomyces cerevisiae has two high affinity sulfate transport systems. Sul3p has been shown to be involved in the transcriptional regulation of the SUL2 gene.

https://www.genetics.org/content/genetics/145/3/627.full.pdf

Molecular characterization of two high affinity sulfate transporters in Saccharomyces cerevisiae.

We have identified a mutation in a gene of Saccharomyces cerevisiae, STR1, that leads to a strict nutritional requirement for cysteine. The str1-1 mutation decreases to an undetectable level the cystathionine gamma-lyase activity. This enzyme catalyzes one of the two reactions involved in the transsulfuration pathway that yields cysteine from homocysteine with the intermediary formation of cystathionine. The phenotype induced by this mutation implies that, in S. cerevisiae, the sulfur atom of sulfide resulting from the reductive assimilation of sulfate is incorporated into a four carbon backbone yielding homocysteine, which, in turn, is the precursor of the biosynthesis of both cysteine and methionine. This also reveals that the direct synthesis of cysteine by incorporation of the sulfur atom into a three carbon backbone as found in Escherichia coli does not occur in S. cerevisiae. The study of the meiotic progeny of diploid strains heterozygous at the STR1 locus has shown that the str1-1 mutation undergoes a particularly high frequency of meiotic gene conversion.

https://www.genetics.org/content/genetics/130/1/51.full.pdf

Genetic analysis of a new mutation conferring cysteine auxotrophy in Saccharomyces cerevisiae: updating of the sulfur metabolism pathway.

Cloning of the MET19 gene revealed that it encodes the glucose-6-phosphate dehydrogenase from yeast. Sequence analysis showed a high degree of similarity between the yeast and the human enzymes. The cloned gene has allowed the construction of a glucose-6-phosphate dehydrogenase null mutant. The only phenotype of such a strain is an absolute requirement for an organic sulfur source, i.e. methionine, S-adenosylmethionine (AdoMet), cysteine, glutathione or homocysteine. The phenotype of this null mutant raises some new questions about the exact functions of the pentose phosphate pathway which was usually considered as the main cellular source of NADPH. Moreover, results reported here show that an increase of the AdoMet pool represses the transcription of the glucose-6-phosphate dehydrogenase gene. This regulation acts on the glucose-6-phosphate dehydrogenase biosynthesis but does not affect the synthesis of 6-phosphogluconate dehydrogenase. That AdoMet controls a part of the pentose phosphate pathway sheds new light on mechanisms regulating the relative fluxes of carbon utilization through the pentose phosphate pathway and glycolysis.

Identification of the structural gene for glucose-6-phosphate dehydrogenase in yeast. Inactivation leads to a nutritional requirement for organic sulfur.

Sulfate uptake is the first step of the sulfate assimilation pathway, which has been shown in our laboratory to be part of the methionine biosynthetic pathway. Kinetic study of sulfate uptake has shown a biphasic curve in a Lineweaver-Burk plot. The analysis of this plot indicates that two enzymes participate in sulfate uptake. One (permease I) has a high affinity for the substrate (Km = 0.005 mM); the other (permease II) shows a much lower affinity for sulfate (Km = 0.35 mM). Regulation of the synthesis of both permeases is under the control of exogenous methionine or S-adenosylmethionine. It was shown, moreover, that synthesis of sulfate permeases is coordinated with the synthesis of the other methionine biosynthetic enzymes thus far studied in our laboratory. An additional specific regulation of sulfate permeases by inhibition of their activity by endogenous sulfate and adenosyl phosphosulfate (an intermediate metabolite in sulfate assimilation) has been shown. A mutant unable to concentrate sulfate has been selected. This strain carried mutations in two independent genes. These two mutations, separated in two different strains, lead to modified kinetics of sulfate uptake. The study of these strains leads us to postulate that there is an interaction in situ between the products of these two genes.

Yolande Surdin-Kerjan

Papers

Metabolism of sulfur amino acids in Saccharomyces cerevisiae.

Molecular characterization of two high affinity sulfate transporters in Saccharomyces cerevisiae.

Genetic analysis of a new mutation conferring cysteine auxotrophy in Saccharomyces cerevisiae: updating of the sulfur metabolism pathway.

Identification of the structural gene for glucose-6-phosphate dehydrogenase in yeast. Inactivation leads to a nutritional requirement for organic sulfur.

Sulfate uptake in Saccharomyces cerevisiae: biochemical and genetic study.