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39890 39896, December 2, 2005 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Karyl I. Minard and Lee McAlister-Henn1 From the Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900
Production of NADPH in Saccharomyces cerevisiae cells grown on glucose has been attributed to glucose-6-phosphate dehydrogenase (Zwf1p) and a cytosolic aldehyde dehydrogenase (Ald6p) (Grabowska, D., and Chelstowska, A. (2003) J. Biol. Chem. 278, 13984 13988). This was based on compensation by overexpression of Ald6p for phenotypes associated with ZWF1 gene disruption and on the apparent lethality resulting from co-disruption of ZWF1 and ALD6 genes. However, we have found that a zwf1 ald6 mutant can be constructed by mating when tetrads are dissected on plates with a nonfermentable carbon source (lactate), a condition associated with expression of another enzymatic source of NADPH, cytosolic NADP -specific isocitrate dehydrogenase (Idp2p). We demonstrated previously that a zwf1 idp2 mutant loses viability when shifted to medium with oleate or acetate as the carbon source, apparently because of the inadequate supply of NADPH for cellular antioxidant systems. In contrast, the zwf1 ald6 mutant grows as well as the parental strain in similar shifts. In addition, the zwf1 ald6 mutant grows slowly but does not lose viability when shifted to culture medium with glucose as the carbon source, and the mutant resumes growth when the glucose is exhausted from the medium. Measurements of NADP(H) levels revealed that NADPH may not be rapidly utilized in the zwf1 ald6 mutant in glucose medium, perhaps because of a reduction in fatty acid synthesis associated with loss of Ald6p. In contrast, levels of NADP rise dramatically in the zwf1 idp2 mutant in acetate medium, suggesting a decrease in production of NADPH reducing equivalents needed both for biosynthesis and for antioxidant functions. tion, a peroxisomal process in yeast that produces hydrogen peroxide in the first reaction of each cycle (5, 6), and acetate is a stringent carbon source that produces rapid flux through mitochondrial respiration, a process associated with the generation of deleterious reactive oxidative species (7, 8). These results suggested that Zwf1p and Idp2p are essential sources of NADPH for thiol-dependent antioxidant functions needed to eliminate detrimental byproducts of oxidative metabolism. Zwf1p expression in yeast is essentially constitutive (1, 2, 9). In contrast, Idp2p levels are repressed by growth on glucose, elevated with growth on nonfermentable carbon sources, and induced during the diauxic shift as glucose is depleted in the medium (9 11). In a search for alternative sources of NADPH in cells grown on glucose, Grabowska and Chelstowska (12) found the ALD6 gene encoding cytosolic NADP -specific acetaldehyde dehydrogenase to be a multicopy suppressor of the Met phenotype of a zwf1 strain. They also reported that a zwf1 ald6 mutant is not viable and concluded that Ald6p is essential for production of NADPH in the absence of Zwf1p. The conclusion that the zwf1 ald6 mutant is not viable (12) was based on the failure of various strategies involving mating and sporulation to obtain this mutant. However, because those experiments were conducted exclusively on glucose medium when Idp2p is not expressed, we postulated that a zwf1 ald6 mutant might be viable if a carbon source conducive for Idp2p expression was utilized during construction. Here we describe construction of the zwf1 ald6 mutant strain and evaluation of the roles of Zwf1p, Idp2p, and Ald6p as enzymatic sources of NADPH.
EXPERIMENTAL PROCEDURES
Cultivation ConditionsYeast strains were cultivated on 2% agar plates or in liquid culture with rich YP medium (1% yeast extract, 2% Bacto-peptone) or with minimal YNB medium (0.17% yeast nitrogen base, 0.5% ammonium sulfate, pH 6.5). Supplements (including methionine for zwf1 mutants) of 20 g/ml were added to minimal medium to satisfy auxotrophic requirements or withheld to select for transformants or diploids. Glucose or lactate as the carbon source was added to 2%. Yeast Strain ConstructionsYeast strains used in this study were isogenic parental haploid MATa and MAT strains (leu23,112 his31 ura357 trp1289; see Ref. 13) and previously constructed mutants of these strains containing deletions and URA3 disruption insertions in the IDP2 or ZWF1 loci (9, 11). For construction of an ALD6 disruption strain, plasmid pFA6a-kanMX4 (14) was used as a template for polymerase chain reaction (PCR) with oligonucleotides (5 -CTATCAGAATACAATGACTAAGCTACACTTTGACACTGCTGAACCAGTCCGTACGCTGCAGGTCGAC and 5 -CTTAATTCTGACAGCTTTTACTTCAGTGTATGCATGGTAGACTTCTTCATCGATGAATTCCAGCTCG) containing sequences from the 5 - and 3 -regions of the ALD6 coding sequence (underlined). The resulting 1.5-kbp DNA fragment was used for targeted gene disruptions in each parental haploid strain and in a MATa strain containing a URA3 disruption of IDP2 (9, 11). Disruption of the ALD6 locus in Kanr transformants was confirmed by Southern blot analysis of EcoRI and EcoRV digests of genomic DNA using radiolabeled probes generated by
Reducing equivalents in the form of NADPH are required for numerous biosynthetic enzymatic reactions and antioxidant mechanisms involving glutathione and/or thioredoxin. The major cellular source of NADPH is thought to be the hexose monophosphate pathway. However, disruption of the Saccharomyces cerevisiae ZWF1 gene encoding glucose-6-phosphate dehydrogenase, the first and rate-limiting enzyme in that pathway, was found to produce relatively mild growth phenotypes including methionine auxotrophy and an increased sensitivity to exogenous oxidizing agents like hydrogen peroxide (1, 2). In studies designed to identify other crucial sources of NADPH in yeast, co-disruption of ZWF1 and the gene (IDP2) encoding cytosolic NADP -specific isocitrate dehydrogenase was found to produce a rapid loss in cell viability following shifts from medium containing glucose to medium containing either oleate or acetate as the carbon source (3, 4). This loss of viability correlated with an increase in levels of endogenous cellular oxidants. Fatty acid metabolism requires rapid flux through -oxida-
* This research was supported by National Institutes of Health Grant AG017477. The
costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence should be addressed: Biochemistry Dept., University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78229-3900. Tel.: 210-567-3782; Fax: 210-567-6595; E-mail: henn@uthscsa.edu.
RESULTS
Construction of a zwf1 ald6 MutantBecause previous attempts to obtain a zwf1 ald6 mutant strain using methods employing glucose as the carbon source were unsuccessful (12), we attempted to construct this strain by mating and sporulation on plates containing a nonfermentable carbon source. For this, we used kanamycin cassette mutagenesis (14) to replace the ALD6 coding region in a haploid yeast strain and confirmed the disruption by Southern blot analysis. This strain was mated with an isogenic strain of opposite mating type containing a URA3 disruption in the coding region of ZWF1 (9). Tetrads were dissected on YP plates containing 2% lactate as the carbon source. The Ura and Kanr markers segregated independently as expected based on the location of ALD6 and ZWF1 genes on different chromosomes.2 In addition, haploid Ura Kanr zwf1 ald6 mutants were obtained at the expected frequency in tetrads dissected on YP lactate plates. Upon replating, the zwf1 ald6 mutants were found to grow well but at slightly reduced rates relative to parental strains on YP plates containing 2% glycerol, lactate, ethanol, or acetate. The mutants did not grow after 7 days on plates containing 2% glucose as expected based on the report by Grabowska and Chelstowska (12). Thus, zwf1 ald6 mutants are viable when constructed and grown on nonfermentable carbon sources. We also constructed a haploid idp2 ald6 strain by transformation of an idp2 strain with the ALD6 kanamycin disruption cassette. In plate tests, we observed no growth phenotypes for the idp2 ald6 strain other than the slow growth on minimal ethanol medium reported previously for an ald6 strain (24). Thus, Idp2p and Ald6p do not appear to have essential overlapping functions. Finally, we attempted to construct a zwf1 ald6 idp2 strain by mating haploid zwf1 ald6 and idp2 strains and by sporulation of diploids on YP lactate plates. However, many of the tetrads from this mating contained only two or three spores. Analysis of the viable spores by tests for Ura and Kanr markers and by Southern blot analysis indicated that mutants containing one or any two of the gene disruptions could be obtained from this mating. However, haploid mutants containing all three gene disruptions were not identified, suggesting that such strains may not be viable. Sources of NADPH for Antioxidant FunctionsTo examine the relative contributions of Zwf1p, Idp2p, and Ald6p as sources of NADPH in protection from endogenous metabolic oxidants, the ald6 and double gene disruption strains (zwf1 ald6 ; idp2 ald6 ; zwf1 idp2 ) (Ref. 9) were shifted from permissive YP lactate medium to YP oleate or YP acetate medium. As shown in Fig. 1, the parental strain () grows well, whereas numbers of viable zwf1 idp2 cells () decrease after 24 h on the new carbon sources. We reported previously (3, 4) that the decrease in viability of the zwf1 idp2 strain following shifts to oleate or acetate medium is coincidental with an increase in levels of cellular oxidants and is presumably due to the loss of critical sources of NADPH needed for cellular antioxidant systems. Both Idp2p and Zwf1p contribute to this function because no loss in viability was observed in similar shifts of mutants lacking only one of these enzymes (3). In contrast to the zwf1 idp2 strain, none of the mutant strains lacking Ald6p, importantly including the zwf1 ald6 strain (f), show any impairment of growth on oleate or acetate (Fig. 1). This suggests that Ald6p is not a crucial source of NADPH for protection from endogenous oxidants generated by peroxisomal -oxidation on oleate medium or by rapid mitochondrial respiration on acetate medium.
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FIGURE 1. Effects of carbon source shifts on viability. Yeast strains were precultured in YP lactate medium and diluted into YP medium with oleate (A) or acetate (B) as the carbon source as described under Experimental Procedures. Viable cell numbers (averages of two or three independent measurements; log scale) are normalized to the starting value, set as 1.0. Yeast strains were: parental (), zwf1 idp2 (), ald6 (), idp2 ald6 (E), and zwf1 ald6 (f).
TABLE ONE
Growth phenotypes with exogenous hydrogen peroxide Yeast strains were streaked onto YP agar plates containing 2% glucose or ethanol as the carbon source and hydrogen peroxide at the indicated concentrations. Colony sizes and numbers, assessed after 7 days of incubation at 30 C, were compared with the growth of each strain on YP plates lacking hydrogen peroxide. , maximal growth; , medium growth; , minimal growth; , no growth.
Strain Parental MATa Parental MAT ald6 idp2 zwf1 ald6 idp2 zwf1 idp2 zwf1 ald6 0 mM H 2O 2 Glucose 3.0 mM 4.0 mM H 2O 2 H 2O 2 5.0 mM H 2O 2 0 mM H 2O 2 1.0 mM H 2O 2 Ethanol 2.0 mM H 2O 2 3.0 mM H 2O 2 4.0 mM H 2O 2
FIGURE 2. Shift of parental () and zwf1 ald6 strains (E) from lactate to glucose medium. Yeast strains were precultured in YP lactate medium and diluted into YP medium with glucose as the carbon source as described under Experimental Procedures. A, viable cell numbers (averages of four or five independent measurements; log scale) are normalized to the starting value, set as 1.0. B, levels of glucose in the medium were determined at the time of cell sampling for viability assays.
FIGURE 3. Expression of Idp2p during shifts from lactate to glucose medium. A, total cellular protein extracts were prepared from the parental and zwf1 ald6 strains prior to (lane 1) and at 12 h (lane 2), 24 h (lane 3), 48 h (lane 4), 72 (lane 5), and 96 h (lane 6) following a representative shift from YP lactate to YP glucose medium as described in the legend for Fig. 2. 25- g samples were used for immunoblot analysis with an antiserum that recognizes both Idp2p as indicated and mitochondrial NADP -specific isocitrate dehydrogenase (the upper band in each lane) (19). B, protein samples from the parental () and zwf1 ald6 strains (E) were used for assays of NADP -specific isocitrate dehydrogenase activity. Activities represent averages of two or three independent determinations.
level. For the zwf1 ald6 strain, immunochemical levels of Idp2p (Fig. 3A) remain repressed during the period of slow growth following the shift to glucose medium (lanes 25 relative to lane 1) and are elevated to parental strain levels at the 96 h time point (lane 6) as glucose is depleted (see Fig. 2). Again, there are corresponding changes in total cellular activity (Fig. 3B, E). Thus, Idp2p levels are elevated in the zwf1 ald6 strain during growth on lactate medium and as glucose levels are reduced at late time points following the culture shift to glucose medium. Changes in Levels of NADP(H) during Carbon Source ShiftsWe measured levels of NADP(H) as described under Experimental Procedures to directly examine correlations between these levels and the loss of viability of a zwf1 idp2 strain following a shift to acetate medium or with the lag in growth of a zwf1 ald6 strain following a shift to glucose medium. Parental and zwf1 idp2 strains were shifted from glucose to acetate medium as described above. Samples taken at various times were used to calculate viable cell numbers and to prepare extracts for measurement of NADP(H) levels (Fig. 4). Prior to the acetate shift, extracts from the parental strain precultivated in glucose medium exhibited levels of 2-fold higher than those of NADPH (time 0 in Fig. 4). FolNADP lowing the shift to acetate medium, levels of NADP () were slightly reduced (by 40%) over the 72-h period, whereas NADPH levels (f) fell by 4-fold in 24 h and remained low. A ratio of [NADP ]:[NADPH] of 6 (Fig. 4B, ) was attained 24 h following the shift and maintained throughout growth to 72 h. For the zwf1 idp2 strain, levels of NADP and NADPH in viable cells were approximately equal prior to the shift (time 0 in Fig. 4), and following the shift to acetate medium, levels of NADPH () dropped in a manner similar to those in the parental strain. In contrast to the parental strain, levels of NADP in the mutant () rose dramatically after 24 h to 5-fold the starting value by 72 h, pro-
ducing a 25-fold increase in the ratio of [NADP ]:[NADPH] (Fig. 4B, E). These results suggest induction of a mechanism in viable zwf1 idp2 cells to produce more NADP perhaps to compensate for the decreased rate of conversion of oxidized to reduced cofactor. Parental and zwf1 ald6 strains were similarly shifted from lactate to glucose medium. Levels of NADP(H) were determined and expressed relative to total cell number in this case (Fig. 5) because both strains remain viable during this shift. Levels of NADP in the parental strain precultivated in lactate medium exceeded those of NADPH by 3.4fold (time 0 in Fig. 5). Following the shift to glucose medium, levels of both NADP and NADPH exhibited a transient increase at 12 h (Fig. 5A), a time corresponding to logarithmic growth and rapid utilization of glucose by the parental strain (Fig. 2), and then declined to near preshift levels after 24 h. The overall ratio of [NADP ]:[NADPH] declined steadily to 1.7 at 48 h (Fig. 5B, ). NADPH levels were below detection in parental strain extracts after this time, which corresponds with stationary phase growth (Fig. 2), precluding estimates of the nucleotide ratio at later time points. The general patterns of changes in levels of NADP and NADPH in the zwf1 ald6 strain shifted from lactate to glucose (Fig. 5A) were quite similar to those for the parental strain, although the peak in the transient increase in levels of both was delayed to 24 h. Also, the ratio of [NADP ]:[NADPH] remained fairly constant ( 2.22.5) (Fig. 5B, E) before and after the shift. The lowest ratio value was obtained 96 h following the shift, a time corresponding to rapid growth of the mutant strain and to depletion of glucose in the medium (Fig. 2). These observations suggest that the dramatic increases in NADP levels and in the ratio of [NADP ]:[NADPH] in the zwf1 idp2 mutant are closely correlated with the loss of viability observed upon a shift of this strain to acetate medium. In contrast, there appears to be little correlation between differences in NADP(H) levels or ratios and the
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FIGURE 4. Levels of NADP(H) in parental and zwf1 idp2 strains shifted to acetate medium. Yeast strains were precultured in YP glucose medium and diluted into YP medium with acetate as the carbon source. A, at the indicated times, viable cell numbers were calculated, and samples of parental (black symbols) and zwf1 idp2 cultures (white symbols) were taken to measure cellular concentrations of NADP (triangles) and NADPH (boxes). B, ratios of the concentrations of NADP / NADPH are indicated for the parental () and zwf1 idp2 strains (E).
FIGURE 5. Levels of NADP(H) in parental and zwf1 ald6 strains shifted to glucose medium. Yeast strains were precultured in YP lactate medium and diluted into YP medium with glucose as the carbon source. A, at the indicated times, viable cell numbers were calculated, and samples of parental (black symbols) and zwf1 ald6 cultures (white symbols) were taken to measure cellular concentrations of NADP (triangles) and NADPH (boxes). B, ratios of the concentrations of NADP / NADPH are indicated for the parental () and zwf1 ald6 strains (E).
slow growth observed for the zwf1 ald6 strain shifted from lactate to glucose medium.
DISCUSSION
We have demonstrated that construction of a haploid zwf1 ald6 mutant is feasible when haploid yeast strains containing the individual gene disruptions are mated, and tetrads from resulting diploids are sporulated on plates containing lactate as the carbon source. In concurrence with a previous report (12), we were unable to obtain the zwf1 ald6 mutant by sporulation of diploids on glucose plates, and the haploid zwf1 ald6 mutant obtained on lactate plates failed to grow when transferred to glucose plates. Grabowska and Chelstowska (12) also demonstrated that multicopy expression of Ald6p or Zms1p, a putative transcriptional regulator of ALD6 expression, can suppress phenotypes associated with ZWF1 disruption. A recent report (25) suggests additional roles for Ald6p and Zwf1p in protection of yeast from
salt stress, a calcineurin-mediated response examined using YP glucose medium containing high concentrations of NaCl or LiCl. Thus, Zwf1p and Ald6p do appear to have an essential overlapping function, presumably production of NADPH, but our results suggest that this function is limited to growth with a fermentable carbon source. Our results also suggest that production of NADPH may not be the sole reason for the glucose phenotype of the zwf1 ald6 strain because levels of reduced cofactor are quite similar in parental and mutant strains following a shift to glucose medium (Fig. 5). Another important function of Ald6p during growth on glucose is provision of acetate as a precursor of cytosolic acetyl-CoA for fatty acid biosynthesis (26). Slow rates of fatty acid synthesis in the zwf1 ald6 strain on glucose medium could directly limit growth and substantially reduce utilization of NADPH reducing equivalents that support fatty acid synthesis. This would reduce rates of interconversion of NADPH and NADP , which, in fact, is what we observe. The ratio of [NADP ]:
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