Hans-Joachim Schüller

Transcriptional control of nonfermentative metabolism in the yeast Saccharomyces cerevisiae

Abstract Although sugars are clearly the preferred carbon sources of the yeast Saccharomyces cerevisiae, nonfermentable substrates such as ethanol, glycerol, lactate, acetate or oleate can also be used for the generation of energy and cellular biomass. Several regulatory networks of glucose repression (carbon catabolite repression) are involved in the coordinate biosynthesis of enzymes required for the utilization of nonfermentable substrates. Positively and negatively acting complexes of pleiotropic regulatory proteins have been characterized. The Snf1 (Cat1) protein kinase complex, together with its regulatory subunit Snf4 (Cat3) and alternative β-subunits Sip1, Sip2 or Gal83, plays an outstanding role for the derepression of structural genes which are repressed in the presence of a high glucose concentration. One molecular function of the Snf1 complex is deactivation by phosphorylation of the general glucose repressor Mig1. In addition to regulation of alternative sugar fermentation, Mig1 also influences activators of respiration and gluconeogenesis, although to a lesser extent. Snf1 is also required for conversion of specific regulatory factors into transcriptional activators. This review summarizes regulatory cis-acting elements of structural genes of the nonfermentative metabolism, together with the corresponding DNA-binding proteins (Hap2-5, Rtg1-3, Cat8, Sip4, Adr1, Oaf1, Pip2), and describes the molecular interactions among general regulators and pathway-specific factors. In addition to the influence of the carbon source at the transcriptional level, mechanisms of post-transcriptional control such as glucose-regulated stability of mRNA are also discussed briefly.

Systematic analysis of yeast strains with possible defects in lipid metabolism

Lipids are essential components of all living cells because they are obligate components of biological membranes, and serve as energy reserves and second messengers. Many but not all genes encoding enzymes involved in fatty acid, phospholipid, sterol or sphingolipid biosynthesis of the yeast Saccharomyces cerevisiae have been cloned and gene products have been functionally characterized. Less information is available about genes and gene products governing the transport of lipids between organelles and within membranes or the turnover and degradation of complex lipids. To obtain more insight into lipid metabolism, regulation of lipid biosynthesis and the role of lipids in organellar membranes, a group of five European laboratories established methods suitable to screen for novel genes of the yeast Saccharomyces cerevisiae involved in these processes. These investigations were performed within EUROFAN (European Function Analysis Network), a European initiative to identify the functions of unassigned open reading frames that had been detected during the Yeast Genome Sequencing Project. First, the methods required for the complete lipid analysis of yeast cells based on chromatographic techniques were established and standardized. The reliability of these methods was demonstrated using tester strains with established defects in lipid metabolism. During these investigations it was demonstrated that different wild‐type strains, among them FY1679, CEN.PK2‐1C and W303, exhibit marked differences in lipid content and lipid composition. Second, several candidate genes which were assumed to encode proteins involved in lipid metabolism were selected, based on their homology to genes of known function. Finally, lipid composition of mutant strains deleted of the respective open reading frames was determined. For some genes we found evidence suggesting a possible role in lipid metabolism. Copyright

The product of the SNF2/SWI2 paralogue INO80 of Saccharomyces cerevisiae required for efficient expression of various yeast structural genes is part of a high-molecular-weight protein complex.

Structural genes of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae are activated by the Ino2p/Ino4p transcription factor that binds to ICRE promoter motifs and mediates maximal gene expression in the absence of inositol. We identified the ino80 mutation causing inositol auxotrophy as a result of a defect in ICRE‐dependent gene activation. The product of the corresponding wild‐type gene INO80 (= YGL150C) shows significant similarity to the Snf2p family of DNA‐dependent ATPases. Nevertheless, SNF2 in increased gene dosage did not suppress ino80 mutant phenotypes. Mutation of the Ino80p lysine residue corresponding to the NTP binding site of Snf2p led to a non‐functional protein. In ino80 null mutants, gene activation mediated by an ICRE decreased to 16% of the wild‐type level. Maximal expression of PHO5, GAL1, CYC1 and ICL1 was also significantly reduced. Thus, Ino80p affects several transcription factors involved in unrelated pathways. As demonstrated by gel filtration, Ino80p is part of a high‐molecular‐weight complex of more than 1 MDa. Similar to what was found for Snf2p, the Ino80p‐containing complex may influence the transcriptional level of several unrelated structural genes by functioning as an ATPase that possibly acts on chromatin.

Transcriptional control of the yeast acetyl‐CoA synthetase gene, ACS1, by the positive regulators CAT8 and ADR1 and the pleiotropic repressor UME6

The ACS1 gene, encoding one out of two acetyl‐CoA synthetase isoenzymes of Saccharomyces cerevisiae, is strictly regulated at the transcriptional level by the carbon source of the medium. While ACS1 is poorly expressed in the presence of a high glucose concentration, a several hundred‐fold derepression occurs with ethanol as the sole carbon source or under conditions of sugar limitation. The molecular mechanism responsible for the carbon source control of ACS1 turned out to be highly complex. A carbon source‐responsive element (CSRE), previously identified upstream of gluconeogenic structural genes, and a binding site of the alcohol dehydrogenase regulator, Adr1p, together mediate about 80% of the derepressed gene activity. Binding of Adr1p synthesized by Escherichia coli to the ACS1 control region was shown by an electrophoretic mobility shift assay. In addition to these activating elements, two URS1 motifs confer negative control on the ACS1 promoter. The URS1 element was found to be a constitutive repression site, which is most effective from a downstream position with respect to an upstream activation site (UAS). In a mutant lacking the URS1‐binding factor, Ume6p, ACS1 expression was partially glucose insensitive. Ume6p must counteract transcription factors that are constitutively active. Site‐directed mutagenesis of Abf1p binding sites in the ACS1 promoter significantly reduced gene expression in the ume6 mutant, grown under repressing conditions. Thus, a functional balance of the pleiotropic positive factor Abf1p and the negative factor Ume6p is in part responsible for glucose repression of ACS1. The combined influence of the regulated UAS elements, CSRE and Adr1p binding site, mediates a strong increase in ACS1 expression under derepressing conditions.

DNA binding site of the yeast heteromeric Ino2p/Ino4p basic helix-loop-helix transcription factor: structural requirements as defined by saturation mutagenesis.

The inositol/choline‐responsive element (ICRE) is an 11 bp cis‐activating sequence motif with central importance for the regulated expression of phospholipid biosynthetic genes in the yeast Saccharomyces cerevisiae. The ICRE containing the CANNTG core binding sequence (E‐box) of basic helix‐loop‐helix (bHLH) regulatory proteins is recognized by the heteromeric bHLH transcription factor Ino2p/Ino4p. In this study, we define the Ino2p/Ino4p consensus binding sequence (5′‐WYTTCAYR‐TGS‐3′) based on the characterization of all possible single nucleotide substitutions. Interestingly, this analysis also identified a single functional deviation (CACATTC) from the CANNTG core recognition element of bHLH proteins. The DNA binding specificities of different yeast bHLH proteins may now be explained by distinct nucleotide preferences especially at two positions immediately preceding the CANNTG core motif.

Carbon source-dependent regulation of the acetyl-coenzyme A synthetase-encoding gene ACSI from saccharomyces cerevisiae

The yeast ACS1 gene, encoding acetyl-coenzyme A synthetase (ACS), was cloned using colony hybridization and a facA probe from Aspergillus nidulans. The complete sequence of 1.5 kb of the ACS1 upstream region was determined. Northern hybridization revealed a strong depression of ACS1 transcripts in a strain grown on the nonfermentable carbon sources, acetate or ethanol. In contrast to a previous report, delta acs1 null mutants did not exhibit a growth defect on acetate medium. Indeed, enzyme assays showed the presence of an additional constitutively expressed ACS activity in delta acs1 mutants. The carbon source-dependent expression was further investigated by the use of an ACS1::lacZ fusion gene, showing complete repression on easily fermentable sugars such as glucose, maltose, sucrose or galactose. Binding sites for the yeast general regulatory factors, Abf1p and Reb1p, together with a sequence reminiscent of the recently identified carbon source-responsive element (CSRE), could be detected in the ACS1 upstream region, presumably mediating the observed regulatory phenotype of this ACS isoenzyme.

Overproduction of the Opi1 repressor inhibits transcriptional activation of structural genes required for phospholipid biosynthesis in the yeast Saccharomyces cerevisiae

Transcription of structural genes required for phospholipid biosynthesis in the yeast Saccharomyces cerevisiae is repressed by high concentrations of inositol and choline. The ICRE (inositol/choline‐responsive element), which is necessary and sufficient for regulation by phospholipid precursors, functions as a binding site for the heterodimeric Ino2/Ino4 activator. ICRE‐dependent transcription becomes constitutive in the absence of the Opi1 repressor. Opi1 contains a leucine zipper motif and two glutamine‐rich stretches. In this work we describe a molecular analysis of OPI1 function and expression. Opi1 mutant variants altered at the leucine zipper and a glutamine‐rich region, respectively, were no longer functional repressors. In contrast, an Opi1 deletion variant lacking the N‐terminal 106 amino acids still mediated negative regulation. Although the leucine zipper suggests that Opi1 may act as a DNA‐binding protein, our data do not support a direct interaction with the ICRE. Despite its function as an antagonist of INO2 and INO4, expression of OPI1 is stimulated by an upstream ICRE. Overexpression of OPI1 under control of the GAL1 promoter severely inhibited activation of ICRE‐dependent genes, leading to inositol‐requiring cells. Growth inhibition of GAL1–OPI1 was observed with INO2 and INO4 alleles activated by either the natural promoter or a heterologous control region. Although induction of GAL1–OPI1 strongly repressed ICRE‐dependent gene expression, the concentration of the Ino2/Ino4 activator remained unchanged. This finding suggests that differential expression of phospholipid biosynthetic genes may occur even in the presence of a constant amount of the specific activator. Copyright

Constitutive expression of yeast phospholipid biosynthetic genes by variants of Ino2 activator defective for interaction with Opi1 repressor

Regulated expression of structural genes involved in yeast phospholipid biosynthesis is mediated by inositol/choline‐responsive element (ICRE) upstream motifs, bound by the heterodimeric activator complex Ino2 + Ino4. Gene repression occurs in the presence of sufficient inositol and choline, requiring an intact Opi1 repressor which binds to Ino2. For a better understanding of interactions among regulators, we mapped an 18 aa repressor interaction domain (RID, aa 118–135) within Ino2 necessary and sufficient for binding by Opi1. By alanine scanning mutagenesis of the entire RID we were able to identify nine residues critical for Opi1‐dependent repression of Ino2 function. Consequently, the corresponding dominant Ino2 variants conferred constitutive expression of an ICRE‐dependent reporter gene and were no longer inhibited even by overproduction of Opi1. Interestingly, Ino2 RID partially overlaps with transcriptional activation domain TAD2. As certain mutations exclusively affect repression while others affect both repression and activation, both functions of Ino2 can be functionally uncoupled. Correspondingly, we mapped the RID–binding activator interaction domain (AID, aa 321–380) at the C‐terminus of Opi1 and introduced missense mutations at selected positions. An Opi1 variant simultaneously mutated at three highly conserved positions showed complete loss of repressor function, confirming RID–AID interaction as the crucial step of regulated expression of ICRE‐dependent genes.

Deregulation of gluconeogenic structural genes by variants of the transcriptional activator Cat8p of the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, growth with a non‐fermentable carbon source requires co‐ordinate transcriptional activation of gluconeogenic structural genes by an upstream activation site (UAS) element, designated CSRE (carbon source‐responsive element). The zinc cluster protein encoded by CAT8 is necessary for transcriptional derepression mediated by a CSRE. Expression of CAT8 as well as transcriptional activation by Cat8p is regulated by the carbon source, requiring a functional Cat1p (= Snf1p) protein kinase. The importance of both regulatory levels was investigated by construction of CAT8 variants with a constitutive transcriptional activation domain (INO2TAD) and/or a carbon source‐independent promoter (MET25 ). Whereas a reporter gene driven by a CSRE‐dependent synthetic minimal promoter showed a 40‐fold derepression with wild‐type CAT8, an almost constitutive expression was found with a MET25–CAT8–INO2TAD fusion construct due to a dramatically increased gene activation under conditions of glucose repression. Similar results were obtained with the mRNA of the isocitrate lyase gene ICL1 and at the level of ICL enzyme activity. Taking advantage of a Cat8p size variant, we demonstrate its binding to the CSRE. Our data show that carbon source‐dependent transcriptional activation by Cat8p is the most important mechanism affecting the regulated expression of gluconeogenic structural genes.

The acetyl‐CoA synthetase gene ACS2 of the yeast Saccharomyces cerevisiae is coregulated with structural genes of fatty acid biosynthesis by the transcriptional activators Ino2p and Ino4p

The yeast Saccharomyces cerevisiae contains two acetyl‐CoA synthetase genes, ACS1 and ACS2. While ACS1 transcription is glucose repressible, ACS2 shows coregulation with structural genes of fatty acid biosynthesis. The ACS2 upstream region contains an ICRE (inositol/choline‐responsive element) as an activating sequence and requires the regulatory genes INO2 and INO4 for maximal expression. We demonstrate in vitro binding of the heterodimeric activator protein Ino2p/Ino4p to the ACS2 promoter. In addition, the pleiotropic transcription factor Abf1p also binds to the ACS2 control region. The identification of ACS2 activating elements also found upstream of ACC1, FAS1 and FAS2 suggests a role of this acetyl‐CoA synthetase isoenzyme for the generation of the acetyl‐CoA pool required for fatty acid biosynthesis.