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Christoph Schüller

Publications and source records attributed to Christoph Schüller.

12 recordsLinked to original sources
PubMed2004

Nuclear localization destabilizes the stress-regulated transcription factor Msn2.

The transcriptional program of yeast cells undergoes dramatic changes during the shift from fermentative growth to respiratory growth. A large part of this response is mediated by the stress responsive transcription factor Msn2. During glucose exhaustion, Msn2 is activated and concentrated in the nucleus. Simultaneously, Msn2 protein levels also drop significantly under this condition. Here we show that the decrease in Msn2 concentration is due to its increased degradation. Moreover, Msn2 levels are also reduced under chronic stress or low protein kinase A (PKA) activity, both conditions that cause a predominant nuclear localization of Msn2. Similar effects were found in msn5 mutant cells that block Msn2 nuclear export. To approximate the effect of low PKA activity on Msn2, we generated a mutant form with alanine substitutions in PKA phosphorylation sites. High expression of this Msn2 mutant is detrimental for growth, suggesting that the increased degradation of nuclear Msn2 might be necessary to adapt cells to low PKA conditions after the diauxic shift or to allow growth under chronic stress conditions.

Active Transport, Cell Nucleus↗
PubMed2004

Expression regulation of the yeast PDR5 ATP-binding cassette (ABC) transporter suggests a role in cellular detoxification during the exponential growth phase.

The yeast ATP-binding cassette transporter Pdr5p mediates pleiotropic drug resistance (PDR) by effluxing a variety of xenobiotics. Immunoblotting demonstrates that Pdr5p levels are high in the logarithmic growth phase, while its levels decrease sharply when cells exit exponential growth. Here, we show that PDR5 promoter activity is dramatically reduced when cells stop growing due to a limitation of glucose or nitrogen or when they approach stationary phase. Interestingly, Pdr3p, a major transcriptional regulator of PDR5, shows the same regulatory pattern. Feeding glucose to starved cells rapidly re-induces both PDR5 and PDR3 transcription. Importantly, diminished Pdr5p levels, as present after starvation, are rapidly restored in response to xenobiotic challenges that activate the transcription factors Pdr1p and Pdr3p. Our data indicate a role for yeast Pdr5p in cellular detoxification during exponential growth.

ATP-Binding Cassette Transporters↗
PubMed2004

Screening the yeast deletant mutant collection for hypersensitivity and hyper-resistance to sorbate, a weak organic acid food preservative.

Certain yeasts cause large-scale spoilage of preserved food materials, partly as a result of their ability to grow in the presence of the preservatives allowed in food and beverage preservation. This study used robotic methods to screen the collection of Saccharomyces cerevisiae gene deletion mutants for both increased sensitivity and increased resistance to sorbic acid, one of the most widely-used weak organic acid preservatives. In this way it sought to identify the non-essential, non-redundant activities that influence this resistance, activities that might be the potential targets of new preservation strategies. 237 mutants were identified as incapable of growth at pH 4.5 in presence of 2 mM sorbic acid, while 34 mutants exhibit even higher sorbate resistance than the wild-type parental strain. A number of oxidative stress-sensitive mutants, also mitochondrial mutants, are sorbate-sensitive. This appears to reflect the importance of sustaining a reducing intracellular environment (high reduced glutathione levels and NADH/NAD and NADPH/NADP ratios). Sorbate resistance is also very severely compromised in mutants lacking an acidified vacuole, in vacuolar protein sorting (vps) mutants, in mutants defective in ergosterol biosynthesis (erg mutants) and with several defects in actin and microtubule organization. Sorbate resistance is, however, elevated with the loss of the Yap5 transcription factor; with single losses of two B-type cyclins (Clb3p, Clb5p); and with loss of a plasma membrane calcium channel activated by endoplasmic reticulum stress (Cch1p/Mid1p).

Drug Resistance, Fungal↗
PubMed2003

Global phenotypic analysis and transcriptional profiling defines the weak acid stress response regulon in Saccharomyces cerevisiae.

Weak organic acids such as sorbate are potent fungistatic agents used in food preservation, but their intracellular targets are poorly understood. We thus searched for potential target genes and signaling components in the yeast genome using contemporary genome-wide functional assays as well as DNA microarray profiling. Phenotypic screening of the EUROSCARF collection revealed the existence of numerous sorbate-sensitive strains. Sorbate hypersensitivity was detected in mutants of the shikimate biosynthesis pathway, strains lacking the PDR12 efflux pump or WAR1, a transcription factor mediating stress induction of PDR12. Using DNA microarrays, we also analyzed the genome-wide response to acute sorbate stress, allowing for the identification of more than 100 genes rapidly induced by weak acid stress. Moreover, a novel War1p- and Msn2p/4p-independent regulon that includes HSP30 was identified. Although induction of the majority of sorbate-induced genes required Msn2p/4p, weak acid tolerance was unaffected by a lack of Msn2p/4p. Ectopic expression of PDR12 from the GAL1-10 promoter fully restored sorbate resistance in a strain lacking War1p, demonstrating that PDR12 is the major target of War1p under sorbic acid stress. Interestingly, comparison of microarray data with results from the phenotypic screening revealed that PDR12 remained as the only gene, which is both stress inducible and required for weak acid resistance. Our results suggest that combining functional assays with transcriptome profiling allows for the identification of key components in large datasets such as those generated by global microarray analysis.

ATP-Binding Cassette Transporters↗
PubMed2003

The CRG1 gene required for resistance to the singlet oxygen-generating cercosporin toxin in Cercospora nicotianae encodes a putative fungal transcription factor.

The Cercospora nicotianae CRG1 gene is involved in cellular resistance to the perylenequinone toxin, cercosporin, that generates highly toxic singlet oxygen upon exposure to light. The entire open reading frame (ORF) of CRG1 was isolated and sequenced. The gene contains an ORF of 1950bp including a 65-bp intron. The predicted 650 amino acid CRG1 protein contains a Cys(6)Zn(2) binuclear cluster DNA-binding motif with homology to various fungal regulatory proteins, indicating that CRG1 may act functionally as a transcription activator. Targeted gene disruption of CRG1 resulted in mutants that are partially sensitive to cercosporin and reduced in cercosporin production. Genetic complementation revealed that CRG1 fully restored cercosporin resistance, but only slightly restored cercosporin production in a UV-derived mutant (CS10) containing a single nucleotide substitution in crg1. Complementation of a crg1-null mutant, however, yielded strains that are similar to the wild-type in both phenotypes. These results indicate that the transcription regulator CRG1 is involved in the activation of genes associated with cercosporin resistance and production in the fungus Cercospora nicotianae.

Amino Acid Sequence↗
PubMed2003

Moderately lipophilic carboxylate compounds are the selective inducers of the Saccharomyces cerevisiae Pdr12p ATP-binding cassette transporter.

Saccharomyces cerevisiae displays very strong induction of a single ATP-binding cassette (ABC) transporter, Pdr12p, when stressed with certain weak organic acids. This is a plasma membrane pump catalysing active efflux of the organic acid anion from the cell. Pdr12p action probably allows S. cerevisiae to maintain lower intracellular levels of several weak organic acid preservatives than would be expected on the basis of the free equilibration of the acid across the cell membrane. This in turn facilitates growth in the presence of these preservatives and therefore yeast spoilage of food materials. Pdr12p appears to confer resistance to those carboxylic acids that, to a reasonable degree, partition into both the lipid bilayer and aqueous phases. Its gene (PDR12) is strongly induced by sorbate, benzoate and certain other moderately lipophilic carboxylate compounds, but not by organic alcohols or high levels of acetate. PDR12 induction reflects the operation of a previously uncharacterized S. cerevisiae stress response, for which the induction signal is probably a high intracellular pool of the organic acid anion.

ATP-Binding Cassette Transporters↗
PubMed2003

The nuclear actin-related protein Act3p/Arp4p of Saccharomyces cerevisiae is involved in transcription regulation of stress genes.

A mutational analysis of the essential nuclear actin-related protein of Saccharomyces cerevisiae, Act3p/Arp4p, was performed. The five residues chosen for substitution were amino acids conserved between actin and Act3p/Arp4p, the tertiary structure of which most probably resembles that of actin. Two thermosensitive (ts) mutants, a single and a double point mutant, and one lethal double point mutant were obtained. Both ts mutants were formamide-sensitive which supports a structural relatedness of Act3p/Arp4p to actin; they were also hypersensitive against hydroxyurea and ultraviolet irradiation pointing to a possible role of Act3p/Arp4p in DNA replication and repair. Their 'suppressor of Ty' (SPT) phenotype, observed with another ts mutant of Act3p/Arp4p before, suggested involvement of Act3p/Arp4p in transcription regulation. Accordingly, genome-wide expression profiling revealed misregulated transcription in a ts mutant of a number of genes, among which increased expression of various stress-responsive genes (many of them requiring Msn2p/Msn4p for induction) was the most salient result. This provides an explanation for the mutant's enhanced resistance to severe thermal and oxidative stress. Thus, Act3p/Arp4p takes an important part in the repression of stress-induced genes under non-stress conditions.

Actins↗
PubMed2003

The yeast protein kinase C cell integrity pathway mediates tolerance to the antifungal drug caspofungin through activation of Slt2p mitogen-activated protein kinase signaling.

The echinocandin caspofungin is a new antifungal drug that blocks cell wall synthesis through inhibition of beta-(1-3)-glucan synthesis. Saccharomyces cerevisiae cells are able to tolerate rather high caspofungin concentrations, displaying high viability at low caspofungin doses. To identify yeast genes implicated in caspofungin tolerance, we performed a genome-wide microarray analysis. Strikingly, caspofungin treatment rapidly induces a set of genes from the protein kinase C (PKC) cell integrity signaling pathway, as well as those required for cell wall maintenance and architecture. The mitogen-activated protein kinase Slt2p is rapidly activated by phosphorylation, triggering signaling through the PKC pathway. Cells lacking genes such as SLT2, BCK1, and PKC1, as well as the caspofungin target gene, FKS1, display pronounced hypersensitivity, demonstrating that the PKC pathway is required for caspofungin tolerance. Notably, the cell surface integrity sensor Wsc1p, but not the sensors Wsc2-4p and Mid2p, is required for sensing caspofungin perturbations. The expression modulation of PKC target genes requires the transcription factor Rlm1p, which controls expression of several cell wall synthesis and maintenance genes. Thus, caspofungin-induced cell wall damage requires Wsc1p as a dedicated sensor to launch a protective response through the activated salvage pathway for de novo cell wall synthesis. Our results establish caspofungin as a specific activator of Slt2p stress signaling in baker's yeast.

Antifungal Agents↗
PubMed2003

War1p, a novel transcription factor controlling weak acid stress response in yeast.

The Saccharomyces cerevisiae ATP-binding cassette (ABC) transporter Pdr12p effluxes weak acids such as sorbate and benzoate, thus mediating stress adaptation. In this study, we identify a novel transcription factor, War1p, as the regulator of this stress adaptation through transcriptional induction of PDR12. Cells lacking War1p are weak acid hypersensitive, since they fail to induce Pdr12p. The nuclear Zn2Cys6 transcriptional regulator War1p forms homodimers and is rapidly phosphorylated upon sorbate stress. The appearance of phosphorylated War1p isoforms coincides with transcriptional activation of PDR12. Promoter deletion analysis identified a novel cis-acting weak acid response element (WARE) in the PDR12 promoter required for PDR12 induction. War1p recognizes and decorates the WARE both in vitro and in vivo, as demonstrated by band shift assays and in vivo footprinting. Importantly, War1p occupies the WARE in the presence and absence of stress, demonstrating constitutive DNA binding in vivo. Our results suggest that weak acid stress triggers phosphorylation and perhaps activation of War1p. In turn, War1p activation is necessary for the induction of PDR12 through a novel signal transduction event that elicits weak organic acid stress adaptation.

ATP-Binding Cassette Transporters↗
PubMed2002

The ATP-binding cassette (ABC) transporter Bpt1p mediates vacuolar sequestration of glutathione conjugates in yeast.

Vacuolar sequestration or cellular extrusion of glutathione-conjugated xenobiotics and catabolites by ATP-binding cassette (ABC) transporters is an important detoxification mechanism operating in many species. In this study, we show that the yeast ABC transporter Bpt1p, a paralogue of Ycf1p, acts as an ATP-dependent vacuolar pump for glutathione conjugates. Bpt1p, which is inhibited by vanadate and glibenclamide, accounts for one third of the total vacuolar transport of glutathione conjugates. Furthermore, immunoblot analyses show that Bpt1p levels are strongly elevated in early stationary phase, consistent with a function of Bpt1p in vacuolar detoxification.

ATP-Binding Cassette Transporters↗
PubMed2002

Acute glucose starvation activates the nuclear localization signal of a stress-specific yeast transcription factor.

In yeast, environmental conditions control the transcription factor Msn2, the nuclear accumulation and function of which serve as a sensitive indicator of nutrient availablity and environmental stress load. We show here that the nuclear localization signal (NLS) of Msn2 is a direct target of cAMP-dependent protein kinase (cAPK). Genetic analysis suggests that Msn2-NLS function is inhibited by phosphorylation and activated by dephosphorylation. Msn2-NLS function is unaffected by many stress conditions that normally induce nuclear accumulation of full-length Msn2. The Msn2-NLS phosphorylation status is, however, highly sensitive to carbohydrate fluctuations during fermentative growth. Dephosphorylation occurs in >2 min after glucose withdrawal but the effect is reversed rapidly by refeeding with glucose. This response to glucose depletion is due to changes in cAPK activity rather than an increase in protein phosphatase activity. Surprisingly, the classical glucose-sensing systems are not connected to this rapid response system. Our results further imply that generic stress signals do not cause short-term depressions in cAPK activity. They operate on Msn2 by affecting an Msn5-dependent nuclear export and/or retention mechanism.

Base Sequence↗