Article Figures & Data
Figures
- FIG. 1.
Comparison of the growth curves of Saccharomyces cerevisiae BY4741 in MM4 medium (open symbols) and in the same basal medium supplemented with a very mildly inhibitory concentration of QN (filled symbols). Growth was followed by measuring the OD600 or by determination of viable cell concentrations, assessed as the number of CFU per ml of cell culture. The results are representative of at least three independent growth experiments. The arrow indicates the time of incubation at which samples for microarray hybridization were harvested.
- FIG. 2.
Functional categorization of genes differentially transcribed in response to QN. Genes showing an activation (upregulated) or repression (downregulated) level equal or greater than twofold after a 15-min treatment with 3.1 mM QN were clustered by functional categories based on the Gene Ontology resource (2) with manual adjustments.
- FIG. 3.
Schematic representation of the data set obtained from the transcriptomic profile of the yeast response to QN (dark bars, upregulated data set; gray bars, downregulated data set). Genes with altered transcription levels were grouped in functional categories according to the GOToolBox database and compared to the entire yeast genome (white bars). A hypergeometric test with Bonferroni correction was performed with a cutoff level of 6 (33). Only categories with statistical significance (P < 0.01) are shown.
- FIG. 4.
Expression of the high-affinity glucose transporter gene HXT7 is activated during the exponential phase of growth with a QN challenge. Yeast cells harboring an HXT7-lacZ fusion plasmid were grown in MM4 medium in the absence (open symbols) or presence (filled symbols) of 3.1 mM QN, and the respective β-galactosidase activities were recorded.
- FIG. 5.
(a) A hypothetical network of transcriptional regulatory associations for the yeast cell response to glucose limitation under QN stress. The regulatory network was based on the microarray results listed in Table S1 in the supplemental material and on the documented regulatory associations between transcription factors and their target genes, as compiled in the YEASTRACT database (www.yeastract.com ) (51). The entire upregulated data set was entered as target genes and grouped according to the respective regulators. Only transcription factors that have been described in the literature as regulators under glucose-limited conditions were included in the model network, together with MTH1 and SNF3, two target genes (white boxes) of the glucose-sensing pathway that are activated in response to QN. Documented associations may be direct, e.g., immunoprecipitation experiments (solid lines), or indirect, e.g., microarray data (dashed lines). The percentage of genes regulated by each transcription factor relative to the total number of genes in the data set is shown. The light-gray boxes indicate transcription factors that are included in the QN-upregulated data set. (b) The growth inhibition phenotype of S. cerevisiae cells induced by QN is reverted upon supplementation of medium with glucose. Suspensions of BY4741 parental strain cells grown to mid-exponential phase in basal medium were serially diluted and spotted on QN-containing MM4 plates, supplemented or not with 1% glucose (for a final 3% concentration). OD600s of cell cultures, from top to bottom, were 0.025, 0.005, and 0.0025.
- FIG. 6.
Deletion of hexose transporter-encoding genes increases yeast tolerance to QN. The susceptibilities to QN of S. cerevisiae parental strain BY4741 cells and of hexose transporter deletion mutants were compared by spot assays. Cellular suspensions grown to mid-exponential phase in basal medium were serially diluted and spotted on QN-containing MM4 plates, supplemented or not with 1% glucose (for a final 3% concentration). OD600s of cell cultures, from left to right, are 0.025, 0.005, and 0.0025.
- FIG. 7.
QN has an inhibitory effect on glucose uptake. The initial rates of uptake of d-[U-14C]glucose were measured in glucose-grown S. cerevisiae cells in the absence (open circles) or presence (filled symbols) of 4.6 mM QN. The drug was either added simultaneously with labeled glucose (filled circles) or preincubated with cells for 5 min (triangles). Plots predicted by computational nonlinear regression (Origin; OriginLab Corporation) are drawn as solid lines. The resulting kinetic parameters are also shown. The data presented are results from at least three independent experiments, each performed in triplicate.
- FIG. 8.
Inhibition of the initial glucose uptake rate by increasing concentrations of QN. Uptake of d-[U-14C]glucose was assayed in the presence of the indicated concentrations of QN. Uptake was measured for 5 s with 25 mM glucose. The resulting curve represents means ± standard errors (<2%), averaged from three independent experiments, each performed in triplicate.
- FIG. 9.
Comparison of the intracellular accumulations of QN in the parental strain and in the hexose transporter deletion mutants. The intracellular concentrations of QN in the different yeast strains were assayed by fluorescence spectroscopy after a 15-min incubation with 4.6 mM QN. The results represent means ± standard errors, averaged from three independent experiments.
Tables
- TABLE 1.
Saccharomyces cerevisiae deletion strains used in this study and obtained from EUROSCARFa
Category and deleted ORF Gene name Gene product function QN susceptibilityb Regulation of metabolism YDR216w ADR1 Carbon source-responsive zinc finger transcription factor — YMR280c CAT8 Transcriptional activator necessary for derepression of genes under nonfermentative growth conditions — YNL027w CRZ1 Transcription factor that activates transcription of genes involved in stress response S++ YPL248c GAL4 Transcription factor required for activation of GAL genes in response to galactose R YDR096w GIS1 Transcription factor involved in expression of genes during nutrient limitation — YHR187w HAP2 Subunit of Elongator complex S YOR358w HAP5 Subunit of heme-activated, glucose-repressed Hap2p/3p/4p/5p CCAAT-binding complex — YBR297w MAL33 MAL activator protein S+ YGL035c MIG1 Transcription factor involved in glucose repression — YGL209w MIG2 Involved in repression, along with Mig1p, of gene expression induced by high levels of glucose S YBR066c NRG2 Transcriptional repressor that mediates glucose repression and negatively regulates filamentous growth R YPR065w ROX1 Heme-dependent repressor of hypoxic genes — YNL167c SKO1 Basic leucine zipper (bZIP) transcription factor S YDR463w STP1 Transcription factor; activates transcription of amino acid permease genes R YHR006w STP2 Transcription factor; activates transcription of amino acid permease genes S++ YIR018w YAP5 Basic leucine zipper (bZIP) transcription factor R Signal transduction YFR014c CMK1 Calmodulin-dependent protein kinase; may play role in stress response — YNL173c MDG1 Plasma membrane protein involved in G-protein-mediated pheromone signaling pathway — YDR277c MTH1 Negative regulator of glucose-sensing signal transduction pathway — YPL026c SKS1 Serine/threonine protein kinase, involved in adaptation to low concentrations of glucose R+ YDL194w SNF3 Plasma membrane glucose sensor that regulates glucose transport R+ Response to stress YPR005c HAL1 Cytoplasmic protein involved in halotolerance — YMR037c MSN2 Transcriptional activator related to Msn4p, activated under stress conditions — YKL062w MSN4 Transcriptional activator related to Msn2p, activated under stress conditions — YDL106c PHO2 Homeobox transcription factor; regulatory targets include genes involved in phosphate metabolism S+ YFR034c PHO4 Basic helix-loop-helix (bHLH) transcription factor of Myc family R YML007w YAP1 Basic leucine zipper (bZIP) transcription factor required for oxidative stress tolerance S+ Membrane transport YCL025c AGP1 Low-affinity amino acid permease with broad substrate range R YHR094c HXT1 Low-affinity glucose transporter R YMR011w HXT2 High-affinity glucose transporter R+ YDR345c HXT3 Low-affinity glucose transporter R+ YHR092c HXT4 High-affinity glucose transporter R YHR096c HXT5 Hexose transporter with moderate affinity for glucose R YJL214w HXT8 Protein of unknown function with similarity to hexose transporter family members R YFL011w HXT10 Putative hexose transporter R YKL217w JEN1 Lactate transporter, required for uptake of lactate and pyruvate — YHL036w MUP3 Low-affinity methionine permease S YML123c PHO84 High-affinity inorganic phosphate (Pi) transporter R YCR037c PHO87 Low-affinity inorganic phosphate (Pi) transporter — YBR106w PHO88 Probable membrane protein, involved in phosphate transport S YBR296c PHO89 Na+/Pi cotransporter, active in early growth phase S+ YFL054c YFL054c Putative channel-like protein — ↵ a Comparative levels of susceptibility for the deletion mutant strains tested are given. Boldface type indicates that the gene was upregulated in response to QN as indicated by microarray analysis.
↵ b —, susceptibility identical to that of the wild-type strain; S, S+, and S++, increased susceptibility levels compared to that of parental strain BY4741. R and R+, decreased susceptibility levels compared to that of parental strain BY4741.
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
-
List of ORFs with twofold reduced or increased expression in response to quinine.
Word file, 205K.
- Supplemental file 1
-
List of ORFs with twofold reduced or increased expression in response to quinine.
