A Dominant Suppressor Mutation of the met30 Cell Cycle Defect Suggests Regulation of the Saccharomyces cerevisiae Met4-Cbf1 Transcription Complex by Met32

Su, Ning-Yuan; Ouni, Ikram; Papagiannis, Christie V.; Kaiser, Peter

doi:10.1074/jbc.m708230200

Cited by 24 publications

(26 citation statements)

References 27 publications

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“…Consistent with our analysis, met32 strains have decreased fitness under sulfate limitation, whereas met31 strains do not (Payen et al 2015). Our data also strengthen the observation that Met31 and Met32 are not fully redundant (Su et al 2008;Cormier et al 2010). Because each binding site in the SUL1 promoter is at least one mismatch away from its consensus site, their annotation by sequence-based motif-finding methods is difficult.…”

Section: Discussionsupporting

confidence: 57%

Comprehensive Analysis of the SUL1 Promoter of Saccharomyces cerevisiae

et al. 2016

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In the yeast Saccharomyces cerevisiae, beneficial mutations selected during sulfate-limited growth are typically amplifications of the SUL1 gene, which encodes the high-affinity sulfate transporter, resulting in fitness increases of .35% . Cis-regulatory mutations have not been observed at this locus; however, it is not clear whether this absence is due to a low mutation rate such that these mutations do not arise, or they arise but have limited fitness effects relative to those of amplification. To address this question directly, we assayed the fitness effects of nearly all possible point mutations in a 493-base segment of the gene's promoter through mutagenesis and selection. While most mutations were either neutral or detrimental during sulfate-limited growth, eight mutations increased fitness .5% and as much as 9.4%. Combinations of these beneficial mutations increased fitness only up to 11%. Thus, in the case of SUL1, promoter mutations could not induce a fitness increase similar to that of gene amplification. Using these data, we identified functionally important regions of the SUL1 promoter and analyzed three sites that correspond to potential binding sites for the transcription factors Met32 and Cbf1. Mutations that create new Met32-or Cbf1-binding sites also increased fitness. Some mutations in the untranslated region of the SUL1 transcript decreased fitness, likely due to the formation of inhibitory upstream open reading frames. Our methodology-saturation mutagenesis, chemostat selection, and DNA sequencing to track variants-should be a broadly applicable approach.KEYWORDS high-throughput sequencing; transcription; promoter mutagenesis; gene amplification; chemostat; yeast; Saccharomyces cerevisiae C HANGES in the extent or timing of gene expression can have profound effects on molecular and organismal phenotypes and thereby drive evolution (King and Wilson 1975;Wray 2007). Heritable noncoding variation can alter gene expression in cis or in trans (Skelly et al. 2009), and both have been shown to contribute significantly to gene expression variation (Ronald et al. 2005;Tirosh et al. 2009;Skelly et al. 2011). Two primary mechanisms by which cis variation can increase gene expression are increases in gene copy number and point mutations in regulatory regions. However, the relative effect size of amplification compared to point mutation is not known.It has been proposed that amplification is both quickly achieved and then reverts after fixation of fitness-increasing point mutations (Hendrickson et al. 2002;Yona et al. 2012). This mechanism raises the question of whether amplification is simply a transitional state that provides an increased chance for a beneficial point mutation to occur. Alternatively, gene amplification may be so frequently observed because the fitness effects it confers are greater than those achievable by point mutations. Gene amplifications have been found to be advantageous in many contexts, including phenotypic evolution (Hoekstra and Coyne 2007;Stern and Orgogozo 2008) an...

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Section: Discussionsupporting

confidence: 57%

Comprehensive Analysis of the SUL1 Promoter of Saccharomyces cerevisiae

et al. 2016

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“…It has previously been demonstrated that Met4 is a substrate for SCF Met30 and a Met30/Met4 complex can be detected in vivo (Kaiser et al, 2000; Rouillon et al, 2000). In addition, Met4 interacts with its co-factor Met32 in vivo and in vitro (Blaiseau and Thomas, 1998; Su et al, 2008), and it was therefore possible that the Met30/Met32 interaction we detected was indirect through the mutual binding partner Met4. Indeed, no interaction between Met30 and Met32 could be detected in met4 Δ mutants, suggesting that Met4 mediates the Met30/Met32 interaction (Figure 2A).…”

Section: Resultsmentioning

confidence: 98%

A Transcriptional Activator Is Part of an SCF Ubiquitin Ligase to Control Degradation of Its Cofactors

2010

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Summary Multi-subunit protein complexes pose a challenge to the coordinated regulation of individual components. We show how the yeast transactivating factor Met4 functions as a component of the SCFMet30 ubiquitin ligase to synchronize its own activity with co-factor assembly. Cells maintain Met4 in a dormant state by a regulatory ubiquitin chain assembled by SCFMet30. Nutritional and heavy metal stress block Met4 ubiquitylation resulting in Met4 activation, which induces a stress response program including cell cycle arrest. Met4 relies on assembly with various co-factors for promoter binding. We report here that the stability of these DNA-binding co-factors is regulated by SCFMet30. Remarkably, the transcriptional activator Met4 functions as a substrate specificity factor in the context of SCFMet30/Met4 to coordinate co-factor degradation with its own activity status. Our results establish an additional layer for substrate recruitment by SCF ubiquitin ligases, and provide conceptual insight into coordinated regulation of protein complexes.

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“…Consistent with Met32 having an important role in MET gene expression, a truncated version of Met32 (Met32D145-192) acts as a dominant suppressor of met30 null mutations by interfering with the recruitment of Met4 to both Cbf1 and Met31/32-dependent promoters (Su et al 2008).…”

Section: Pathway Genesmentioning

confidence: 99%

Regulation of Amino Acid, Nucleotide, and Phosphate Metabolism in Saccharomyces cerevisiae

2012

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Ever since the beginning of biochemical analysis, yeast has been a pioneering model for studying the regulation of eukaryotic metabolism. During the last three decades, the combination of powerful yeast genetics and genome-wide approaches has led to a more integrated view of metabolic regulation. Multiple layers of regulation, from suprapathway control to individual gene responses, have been discovered. Constitutive and dedicated systems that are critical in sensing of the intra- and extracellular environment have been identified, and there is a growing awareness of their involvement in the highly regulated intracellular compartmentalization of proteins and metabolites. This review focuses on recent developments in the field of amino acid, nucleotide, and phosphate metabolism and provides illustrative examples of how yeast cells combine a variety of mechanisms to achieve coordinated regulation of multiple metabolic pathways. Importantly, common schemes have emerged, which reveal mechanisms conserved among various pathways, such as those involved in metabolite sensing and transcriptional regulation by noncoding RNAs or by metabolic intermediates. Thanks to the remarkable sophistication offered by the yeast experimental system, a picture of the intimate connections between the metabolomic and the transcriptome is becoming clear.

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A Dominant Suppressor Mutation of the met30 Cell Cycle Defect Suggests Regulation of the Saccharomyces cerevisiae Met4-Cbf1 Transcription Complex by Met32

Cited by 24 publications

References 27 publications

Comprehensive Analysis of the SUL1 Promoter of Saccharomyces cerevisiae

Comprehensive Analysis of the SUL1 Promoter of Saccharomyces cerevisiae

A Transcriptional Activator Is Part of an SCF Ubiquitin Ligase to Control Degradation of Its Cofactors

Regulation of Amino Acid, Nucleotide, and Phosphate Metabolism in Saccharomyces cerevisiae

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