W. Scott Moye-Rowley

The Response to Heat Shock and Oxidative Stress in Saccharomyces cerevisiae

A common need for microbial cells is the ability to respond to potentially toxic environmental insults. Here we review the progress in understanding the response of the yeast Saccharomyces cerevisiae to two important environmental stresses: heat shock and oxidative stress. Both of these stresses are fundamental challenges that microbes of all types will experience. The study of these environmental stress responses in S. cerevisiae has illuminated many of the features now viewed as central to our understanding of eukaryotic cell biology. Transcriptional activation plays an important role in driving the multifaceted reaction to elevated temperature and levels of reactive oxygen species. Advances provided by the development of whole genome analyses have led to an appreciation of the global reorganization of gene expression and its integration between different stress regimens. While the precise nature of the signal eliciting the heat shock response remains elusive, recent progress in the understanding of induction of the oxidative stress response is summarized here. Although these stress conditions represent ancient challenges to S. cerevisiae and other microbes, much remains to be learned about the mechanisms dedicated to dealing with these environmental parameters.

A nuclear receptor-like pathway regulating multidrug resistance in fungi

Multidrug resistance (MDR) is a serious complication during treatment of opportunistic fungal infections that frequently afflict immunocompromised individuals, such as transplant recipients and cancer patients undergoing cytotoxic chemotherapy. Improved knowledge of the molecular pathways controlling MDR in pathogenic fungi should facilitate the development of novel therapies to combat these intransigent infections. MDR is often caused by upregulation of drug efflux pumps by members of the fungal zinc-cluster transcription-factor family (for example Pdr1p orthologues). However, the molecular mechanisms are poorly understood. Here we show that Pdr1p family members in Saccharomyces cerevisiae and the human pathogen Candida glabrata directly bind to structurally diverse drugs and xenobiotics, resulting in stimulated expression of drug efflux pumps and induction of MDR. Notably, this is mechanistically similar to regulation of MDR in vertebrates by the PXR nuclear receptor, revealing an unexpected functional analogy of fungal and metazoan regulators of MDR. We have also uncovered a critical and specific role of the Gal11p/MED15 subunit of the Mediator co-activator and its activator-targeted KIX domain in antifungal/xenobiotic-dependent regulation of MDR. This detailed mechanistic understanding of a fungal nuclear receptor-like gene regulatory pathway provides novel therapeutic targets for the treatment of multidrug-resistant fungal infections.

Expression of a Glutamate Decarboxylase Homologue Is Required for Normal Oxidative Stress Tolerance in Saccharomyces cerevisiae

The action of γ-aminobutyrate (GABA) as an intercellular signaling molecule has been intensively studied, but the role of this amino acid metabolite in intracellular metabolism is poorly understood. In this work, we identify a Saccharomyces cerevisiae homologue of the GABA-producing enzyme glutamate decarboxylase (GAD) that is required for normal oxidative stress tolerance. A high copy number plasmid bearing the glutamate decarboxylase gene (GAD1) increases resistance to two different oxidants, H2O2 and diamide, in cells that contain an intact glutamate catabolic pathway. Structural similarity of the S. cerevisiae GAD to previously studied plant enzymes was demonstrated by the cross-reaction of the yeast enzyme to a antiserum directed against the plant GAD. The yeast GAD also bound to calmodulin as did the plant enzyme, suggesting a conservation of calcium regulation of this protein. Loss of either gene encoding the downstream steps in the conversion of glutamate to succinate reduced oxidative stress tolerance in normal cells and was epistatic to high copy number GAD1. The gene encoding succinate semialdehyde dehydrogenase (UGA5) was identified and found to be induced by H2O2 exposure. Together, these data strongly suggest that increases in activity of the glutamate catabolic pathway can act to buffer redox changes in the cell.

Transcriptional activation by the SV40 AP-1 recognition element in yeast is mediated by a factor similar to AP-1 that is distinct from GCN4

The consensus recognition element for the mammalian transcription factor AP-1 is very similar to that of the transcriptional activator GCN4. Here, we show that the AP-1 recognition element (ARE) found in the SV40 enhancer can activate transcription from a heterologous promoter in S. cerevisiae. This activation, however, is not dependent on the presence of GCN4 as evidenced by ARE-dependent transcription in a gcn4 yeast strain. A previously unknown yeast transcription factor that is probably responsible for this activation was identified and highly purified. The yeast factor, designated yAP-1, shares remarkably similar biochemical and DNA-binding characteristics with mammalian AP-1. These data suggest that the yeast and mammalian AP-1 are evolutionarily conserved and perhaps functionally related. Also note-worthy is that GCN4 can bind to a GCN4 recognition element (GCRE) and to the ARE with approximately equal affinities; yAP-1, however, has a much lower affinity for the GCRE than the ARE, suggesting that yAP-1 can discriminate between these elements in vivo.

Analysis of the oxidative stress regulation of the Candida albicans transcription factor, Cap1p.

CAP1 encodes a basic region‐leucine zipper (bZip) transcriptional regulatory protein that is required for oxidative stress tolerance in Candida albicans. Cap1p is a homologue of a Saccharomyces cerevisiae bZip transcription factor designated Yap1p that is both required for oxidative stress tolerance and localized to the nucleus in response to the presence of oxidants. Oxidant‐regulated localization of Yap1p to the nucleus requires the presence of a carboxy‐terminal cysteine residue (C629) that is conserved in Cap1p as C477. To examine the role of this conserved cysteine residue, C477 was replaced with an alanine residue. This mutant protein, C477A Cap1p, was analysed for its behaviour both in S. cerevisiae and C. albicans. Wild type and C477A Cap1p were able to complement the oxidant hypersensitivity of a Δyap1 S. cerevisiae strain. Whereas a Yap1p‐responsive lacZ fusion gene was oxidant inducible in the presence of YAP1, the C. albicans Cap1p derivatives were not oxidant responsive in S. cerevisiae. Introduction of wild type and C477A Cap1p‐expressing plasmids into C. albicans produced differential resistance to oxidants. Glutathione reductase activity was found to be inducible by oxidants in the presence of Cap1p but was constitutively elevated in the presence of C477A Cap1p. Western blot assays indicate Cap1p is post‐translationally regulated by oxidants. Green fluorescent protein fusions to CAP1 showed that this protein is localized to the nucleus only in the presence of oxidants while C477A Cap1p is constitutively nuclear localized. Directly analogous to S. cerevisiae Yap1p, regulated nuclear localization of C. albicans Cap1p is crucial for its normal function.

Regulation of the Transcriptional Response to Oxidative Stress in Fungi: Similarities and Differences

Fungal cells must deal with a wide variety of potentially toxic environmental challenges during the course of their proliferation. Often these environmental changes are designed to target and eliminate the fungus along with other microorganisms from a specific milieu, such as an animal host. An

Multidrug Resistance in Fungi

One of the major advances serving to define the beginning of the era of modern medicine was the development of penicillin in the early 1940s as the first widely used antibiotic effective against microorganisms (reviewed in reference [111][1]). In what has become an all-too-common trend, antibiotic-

Transcriptional Control of Multidrug Resistance in the Yeast Saccharomyces

A major problem in chemotherapeutic treatment of many pathological conditions including cancer and fungal infections is the development of a multidrug-resistant state in the target cell. Saccharomyces cerevisiae cells can be isolated that have single genetic alterations that cause the resulting mutant strains to become tolerant of a wide range of compounds that would otherwise be toxic. These mutant cells are referred to as having a pleiotropic drug-resistant (Pdr) phenotype. Studies of these Pdr cells have demonstrated that mutations either within genes encoding transcriptional regulators or in their regulatory inputs lead to overexpression of downstream transporter proteins with associated multidrug resistance. This review is aimed at providing a framework for understanding the networks modulating expression of PDR genes in S. cerevisiae.

New Insights into the Pleiotropic Drug Resistance Network from Genome-Wide Characterization of the YRR1 Transcription Factor Regulation System

ABSTRACT Yrr1p is a recently described Zn2Cys6 transcription factor involved in the pleiotropic drug resistance (PDR) phenomenon. It is controlled in a Pdr1p-dependent manner and is autoregulated. We describe here a new genome-wide approach to characterization of the set of genes directly regulated by Yrr1p. We found that the time-course production of an artificial chimera protein containing the DNA-binding domain of Yrr1p activated the 15 genes that are also up-regulated by a gain-of-function mutant of Yrr1p. Gel mobility shift assays showed that the promoters of the genes AZR1, FLR1, SNG1, YLL056C, YLR346C, and YPL088W interacted with Yrr1p. The putative consensus Yrr1p binding site deduced from these experiments, (T/A)CCG(C/T)(G/T)(G/T)(A/T)(A/T), is strikingly similar to the PDR element binding site sequence recognized by Pdr1p and Pdr3p. The minor differences between these sequences are consistent with Yrr1p and Pdr1p and Pdr3p having different sets of target genes. According to these data, some target genes are directly regulated by Pdr1p and Pdr3p or by Yrr1p, whereas some genes are indirectly regulated by the activation of Yrr1p. Some genes, such as YOR1, SNQ2, and FLR1, are clearly directly controlled by both classes of transcription factor, suggesting an important role for the corresponding membrane proteins.

Mutational Disruption of Plasma Membrane Trafficking of Saccharomyces cerevisiae Yor1p, a Homologue of Mammalian Multidrug Resistance Protein

ABSTRACT The ATP binding cassette (ABC) transporter protein Yor1p was identified on the basis of its ability to elevate oligomycin resistance when it was overproduced from a high-copy-number plasmid. Analysis of the predicted amino acid sequence of Yor1p indicated that this protein was a new member of a subfamily of ABC transporter proteins defined by the multidrug resistance protein (MRP). In this work, Yor1p is demonstrated to localize to the Saccharomyces cerevisiaeplasma membrane by both indirect immunofluorescence and biochemical fractionation studies. Several mutations were generated in the amino-terminal nucleotide binding domain (NBD1) of Yor1p to test if the high degree of sequence conservation in this region of the protein was important for function. Deletion of a phenylalanine residue at Yor1p position 670 led to a mutant protein that appeared to be retained in the endoplasmic reticulum (ER) and that was unstable. As shown by others, deletion of the analogous residue from a second mammalian MRP family member, the cystic fibrosis transmembrane conductance regulator (CFTR), also led to retention of this normally plasma membrane-localized protein in the ER. Changes in the spacing between or the sequences flanking functional motifs of Yor1p NBD1 led to defective trafficking or decreased activity of the mutant proteins. Analyses of the degradation of wild-type and ΔF670 Yor1p indicated that the half-life of ΔF670 Yor1p was dramatically shortened. While the vacuole was the primary site for turnover of wild-type Yor1p, degradation of ΔF670 Yor1p was found to be more complex with both proteasomal and vacuolar contributions.