The GLO1 Gene Is Required for Full Activity of O ... - ACS Publications

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The GLO1 Gene is Required for Full Activity of OAcetyl Homoserine Sulfhydrylase encoded by MET17 Matias I. Kinzurik, Kien Ly, Karine M. David, Richard C. Gardner, and Bruno Fedrizzi ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00815 • Publication Date (Web): 09 Dec 2016 Downloaded from http://pubs.acs.org on December 17, 2016

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ACS Chemical Biology

The GLO1 Gene is Required for Full Activity of O-Acetyl Homoserine Sulfhydrylase encoded by MET17

Matias I. Kinzurik

§,*

, Kien Ly †, Karine M. David †, Richard C. Gardner †, and Bruno

Fedrizzi §,*

§

School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland, New

Zealand †

School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New

Zealand

* Author to whom correspondence should be addressed: Tel: + 64 9 9238473 Fax: + 64 9 3737422 E-mail: [email protected] (MIK); [email protected] (BF).

ACS Paragon Plus Environment


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Abstract. During glycolysis, yeast generates methylglyoxal (MG), a toxic metabolite that affects growth. Detoxification can occur when glyoxylase I (GLO1) and glyoxylase II (GLO2) convert MG to lactic acid. We have identified an additional, previously unrecognized role for GLO1 in sulfur assimilation in the yeast Saccharomyces cerevisiae. During a screening for putative carbon-sulfur lyases, the glo1 deletion strain showed significant production of H2S during fermentation. The glo1 strain also assimilated sulfate inefficiently, but grew normally on cysteine. These phenotypes are consistent with reduced activity of the O-acetyl homoserine sulfhydrylase, Met17p. Overexpression of Glo1p gave a dominant negative phenotype that mimicked the glo1 and met17 deletion strain phenotypes. Western analysis revealed reduced expression of Met17p in the glo1 deletion, but there was no indication of an altered conformation of Met17p or any direct interaction between the two proteins. Unravelling a novel function in sulfur assimilation and H2S generation in yeast for a gene never connected with this pathway provides new opportunities for the study of this molecule in cell signalling, as well as the potential regulation of its accumulation in the wine and beer industry.

Keywords Saccharomyces cerevisiae, hydrogen sulfide, MET17, sulfate assimilation, methylglyoxal.


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Introduction

Hydrogen sulfide (H2S) is a highly volatile sulfur compound that has recently emerged as an important signalling molecule. Like nitric oxide, and carbon monoxide, H2S plays critical roles in cellular signalling and hormonal regulation.1-3 Additionally, H2S is produced by yeast as a particularly undesirable consequence of grape juice fermentation during winemaking. It is commonly associated with off-flavours often described as reminiscent of rotten egg and putrefaction.4, 5 In Saccharomyces cerevisiae, H2S arises mostly as an intermediate of the sulfate assimilation pathway (SAP), where it is produced by the action of the sulfite reductase complex, Met5p/Met10p.6 O-acetylhomoserine sulfhydrylase, Met17p, then catalyses the reaction of H2S with O-acetyl homoserine to produce homocysteine, which is further incorporated into the sulfur-containing amino acids and glutathione7 (Fig. 1A). Other sources of H2S production include the degradation of sulfur-containing amino acids7 and the reduction of elemental sulfur.8-9 Genetic changes that result in the inefficient incorporation of reduced sulfur into these end products are believed to lead to the accumulation of H2S.10-13 Several synthetic biology strategies to reduce H2S accumulation have been attempted in yeast including the overexpression of the Met17p,11 the use of null or very low activity met5 or met10 mutants,10, 12

and the expression of a more active MET2 gene; the latter was presumed to produce more

of the co-substrate O-acetyl homoserine, that could be then taken up by MET17, consuming in turn a proportional number of H2S molecules (Fig. 1A).13 During glycolysis, yeast generates MG, a toxic natural metabolite that affects growth.14 Detoxification occurs when Glyoxylase I (encoded by GLO1) converts MG to S-Dlactoylglutathione in the presence of glutathione (GSH). Subsequently, Glyoxylase II



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(encoded by GLO2), or its mitochondrially targeted homolog, Glyoxylase IV (encoded by GLO4), further hydrolyses this glutathione thiol ester to lactic acid and glutathione 15-17 (Fig. 1B). The rapid catalysis of MG seems to be critical for yeast, and additional detoxification pathways that are GSH-independent have appeared throughout evolution.18, 19 Although, to the best of our knowledge, GLO1 is not known to be associated with the SAP, the enzyme has been found to have sequence similarities with known carbon-sulfur lyases, 20 pinpointing a possible role in sulfur metabolism. In our laboratory, a preliminary screening of fermentations with putative carbon-sulfur lyase knockout strains in synthetic grape juice media showed a significant production of H2S in a strain deleted for GLO1. This finding led us to investigate whether the methylglyoxal pathway genes have a role in the formation of H2S.

Results and Discussion

A glo1 deletion mutant produces H2S, but not a glo2 or glo4 deletion

During a screening for putative carbon-sulphur lyase genes, ferments were performed in synthetic medium resembling grape juice and assessed for H2S production using detection columns. Monitoring weight loss showed that all of the deletion strains fermented at similar rates (Fig. S1). As reported previously,21 fermentation with the deletion mutant of glo1 (YML004C) was found to release H2S to the point of saturating the detection limit of the detection column within 24 h, while the wild-type yeast strain BY4743 produced no detectable levels of the gas (Table 1). In similar fashion, two positive control strains with deletions in either MET17


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(YLR303W) gene -which is unable to catalyse the addition of H2S to O-acetyl-homoserine-, or MET2 (YNL277W), -which is unable to produce O-acetyl homoserine- accumulated H2S as expected (Fig. 1A). The other GSH-dependent methylglyoxal pathway genes were also screened for H2S production. Ferments with deletions in GLO2 (YDR272W) or GLO4 (YOR040W) did not produce any detectable levels of H2S, like the wild type strain, hinting at a possible genespecific effect for this phenotype.

H2S accumulated by glo1 mutants comes from sulfate assimilation

Figure 2A shows that when the glo1 and met17 deletion mutants were fermented in small volumes of SGM to quantify their total H2S production, both strains showed equivalent production. Synthetic grape media 22 contains four major sources of sulfur: methionine (0.15 mM), cysteine (0.4 mM), glutathione (0.67 mM) and sulfate (5 mM). In order to establish which of these might be the precursor responsible for H2S accumulation in glo1 mutants, 20mL ferments were performed in SGM with each of the sulfur-containing nutrients added individually (at the concentration found in SGM). The glo1 mutant produced H2S only when supplemented with 5 mM sulfate (Fig. 2A), and no production of this gas was detected when methionine, cysteine or glutathione were used as sole sulfur sources. Regular SGM media produced less H2S than that with only sulfate as a sulfur source. Presumably the presence of cysteine, methionine and GSH exerts negative feedback on the sulfur assimilation pathway to reduce the flow of metabolites, consistent with previous reports of feedback inhibition and tight transcriptional regulation of SAP genes.23, 24 In media with only sulfate, any inability to assimilate H2S to homocysteine would be predicted to lead to a shortage of sulfur-containing



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amino acids, and therefore be a metabolic trigger for even more of this gas accumulating as the pathway tries to correct itself. The met17 deletion mutant also showed no H2S production when grown with methionine or cysteine alone, and did not grow (as expected) when the media contained sulfate as the only sulfur source (data not shown). These results suggested that the glo1 mutant produces H2S as a result of altered metabolite flow down the SAP. To confirm this conclusion, the met17 and glo1 deletion mutants were fermented using a low concentration of methionine to allow growth (0.075 mM), with additional sulfate added. For both strains, the production of H2S increased with increasing sulfate in the medium, as shown in Figure 2B.

At low and

intermediate sulfate concentrations, H2S production from the met17 mutant was approximately double that from the glo1 deletion strain, but their total production was indistinguishable at 5 mM sulfate. The wild type BY4743, again showed no production of H2S at any sulfate concentration.

Methylglyoxal accumulation does not induce H2S production

GLO1 is known to catalyse the condensation of glutathione with methylglyoxal (MG) to give S-D-lactoylglutathione

15

(Fig. 1B). The deletion of this gene was previously shown to

increase MG concentration inside the cell, making it more susceptible to toxicity by external addition of this glycolysis by-product.25 MG production is a consequence of glycolysis, and the toxicity of this compound has led to the evolution of multiple biosynthetic pathways devoted to quench this molecule’s detrimental effects, including multiple glyoxylase genes in yeast.26 Moreover, MG toxicity is well studied in mammals where it has been shown to be involved in diabetes and age-related diseases, 27 but the exact mechanism of toxicity in yeast is not yet known. Furthermore, it was


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found that MG regulates H2S release in rat vascular smooth muscle cells by directly reacting with H2S or inducing gene expression that leads to the production of H2S.28 To establish whether an increase in methylglyoxal concentration levels in yeast cells could be responsible for the production of the H2S detected, five knockout strains of known GSHindependent glyoxylases

18, 19, 29

were selected from the BY4743 collection and grown on

indicator plates (BiYNB) 7, 10 containing minimal media with added bismuth to monitor H2S production, and containing 0.2%, 2% or 20% glucose to induce glycolysis 30 (Fig. 3). Yeast is known to ferment glucose at concentrations that exceed 0.5% (w/v), regardless of being in aerobiosis, 31 thus leading to the accumulation of glycolysis metabolites, including MG. Dark colony coloration, indicating production of H2S, was observed only for the GLO1 knockout, and the two positive controls: knockout strains in MET2 and MET17. Singledeletion mutants of other glyoxylases analyzed tested negative for H2S production, similar both to the wild-type strain and to the negative control knockout in MET10, which blocks sulfate assimilation at bisulfite production (Fig. 1A). Furthermore, the MG sensitivity of the glo1 deletion strain was not affected by the concentration of sulfate in SGM (Fig. S2). We concluded that H2S production by the glo1 deletion strain is not linked to methylglyoxal accumulation, based on the lack of coloration in the mutant glyoxylase strains, the lack of difference in coloration between met17 and glo1 deletion strains, and the fact that MG sensitivity for this strain remains, independently of whether H2S is produced or not. Furthermore, addition of methylglyoxal at 0.1 mM, 1 mM, 5 mM or 10 mM concentration in SGM fermentations, using either BY4743 or these mutant strains, produced no detectable levels of H2S (data not shown).

A glo1 deletion can only partially assimilates sulfate because of a MET17 deficiency



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H2S accumulation from sulfate assimilation can be brought about by reduced activity of two pathways 7 : one is the homoserine biosynthetic pathway, including the HOM6, HOM2 and HOM3 genes that produce homoserine, and the MET2 gene that produces L-homoserine Oacetyl transferase; the other pathway is the SAP, which includes the O-acetyl homoserine Oacetyl serine sulfhydrylase gene, MET17. A disruption in the expression of any of these five genes results in no or inefficient synthesis of homocysteine, and the subsequent sulfur containing amino acids,

10

along with a consequent build-up of H2S.7 In contrast, deletion

mutations in genes above MET17 in the SAP (MET3, -14, -16, -5 and -10, see Fig. 1), all result in reduced levels of H2S production.7 However, it is also possible that the H2S accumulation could result from up-regulation of one or more of the steps upstream in the pathway. To identify how the SAP was affected in the glo1 deletion mutant, the growth rates of met17, met2, and glo1 deletion strains were monitored in SGM media containing limiting concentrations of either methionine, cysteine or sulfate, compared to the wild-type BY4743 strain. It should be noted that met2 mutants (like hom6, -2 and -3 mutants) are unable to grow on cysteine as a sole sulfur source because the trans-sulfuration pathway is inactive.32 Met2p catalyses the formation of O-acetyl homoserine from homoserine and acetyl CoA.33 In addition to acting as a substrate for Met17p, O-acetyl homoserine is required as a substrate by the cystathionine β-synthase, STR2,

34

the first step in the conversion of cysteine to

methionine (see Fig. 1). Therefore, a met2 mutant cannot synthesise methionine in media containing cysteine as a sole sulfur source,

32

and so is auxotrophic for both sulfate and

cysteine. The growth data for BY4743 and the met17, met2 and glo1 mutants are shown in Figure 4. For BY4743, maximal growth rates and final cell titres were obtained with 75 µM cysteine or 37.5 µM methionine, but were not quite attained at the highest concentration of sulfate used


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(150 µM). All three deletion strains grew well on methionine. As expected, the strains deleted for met2 and met17 could not grow with sulfate as the only sulfur source, nor could met2 in media containing only cysteine. The glo1 deletion strain behaved similarly to the wild type strain in increasing concentrations of both methionine and cysteine, but showed partial growth retardation in media where sulfate was the only sulfur source. Even at the highest molarity of sulfate used, this strain was unable to match BY4743, with a slower µmax and a lower final cell titre (assessed by OD600). The growth rates in glutathione showed no difference to those in cysteine, and all strains grew normally in regular SGM (Fig. S3). The poor growth of the glo1 mutant on sulfate is consistent with this strain having restricted metabolite flow down the SAP, rather than any upregulation. Its wild-type growth rate on cysteine, in contrast to the met2 deletion, shows that this strain is able to synthesise O-acetylhomoserine normally, and is therefore not deficient in the homoserine arm of the SAP (Fig 1a). Based on these growth data and the production of H2S, we concluded that the glo1 mutant appears to be partly defective in the formation of homocysteine from H2S and Oacetyl homoserine, a step that is known to require Met17p. To test this idea, a met17-glo1 double mutant was fermented in SGM media, and shown to produce similar amounts of H2S to that of the met17 single mutant (Fig. S4), consistent with the conclusion that the H2S producing phenotype in glo1 is due to a disruption in the same branch of the pathway as the MET17 enzyme.

Glo1p overexpression also results in sulfate assimilation deficiency and H2S accumulation

We introduced pE24GLO1, a GLO1 overexpression vector,

15

into BY4743 and the glo1

deletion mutant, in order to observe complementation. We evaluated growth rate and H2S production as before. The data are shown in Figure 5A.



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Surprisingly, overexpression of GLO1 in a wild-type BY4743 strain resulted in partial growth retardation in media where sulfate was the only sulfur source. In addition, the overexpressing strain gave elevated H2S production in bismuth-containing indicator plates with minimal media (Fig. 5B). Complementation in the glo1 deletion strain with the pE24GLO1, Glo1poverexpressing, plasmid was also not achieved. This strain showed even lower growth rates in 0.15 mM sulfate containing media and no apparent decrease in coloration of the colony in the H2S detection plate was detected when compared to the single mutant (Fig. 5A and 5B). These results confirm that altering the expression of GLO1 affects both growth on sulfate and H2S production; however, the phenotypes observed from overexpression were the same as those seen in the deletion mutant. Furthermore, the Glo1p-overexpressing plasmid was also introduced into a met17 mutant and its H2S accumulation rate compared to that of the single met17 deletion strain and that of the met17-glo1 double deletion strain resulting in no differences found (Fig. S4). These results together indicate that overexpression of Glo1p has a dominant negative effect on the activity of O-acetyl homoserine sulfhydrylase, encoded by MET17.

A glo1 mutant shows reduced expression of Met17p

Dominant negative effects like the one observed are often seen when two proteins physically interact to form an active multimer.35 Therefore, we attempted to identify direct interactions between the Glo1p and Met17p. Like many enzymes in the aminotransferase family, Met17p is multimeric, with the active enzyme in yeast reported to be a tetramer based on its size on denaturing gels.36 Heterologous expression of MET17 in E. coli showed that this enzyme forms a homotetramer that possesses O-acetyl serine hydrolase activity.37 However, it seemed possible that, in vivo in yeast, one or


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more Glo1p subunits are incorporated into a Met17p-Glo1p hetero-tetramer, and provide activity that is enhanced over that of a purely Met17p tetramer. We therefore performed a pull-down assay using whole protein extract from a hybrid BY4743-like strain containing integrated copies of a Met17p-GFP fusion protein and a TAPtagged Glo1p. Anti-GFP antibodies was bound to agarose beads and incubated with whole cell extract. The subsequent eluent fraction was assessed for the presence of TAP tag among those protein that interacted with Met17p-GFP. However, no evidence for a direct interaction between Met17p and Glo1p could be observed in this study (Fig. S5A). In addition, a native polyacrylamide gel (PAGE) followed by a western blot to identify yeast strains expressing Met17p-GFP in either a wild type, glo1 deletion, or Glo1p overexpression background failed to demonstrate differences in the electrophoresed distance for this fusion protein (Fig. S5B), suggesting no change in multimer conformation as a result of changes in the glo1 phenotype of the strain. However, the concentration of Met17p appeared to differ between strains in this Western blot. Protein quantification for Met17p-GFP was therefore undertaken using SDS-PAGE for these same three strains. The data confirmed a significant drop (p < 0.05) in this fusion protein’s expression in the glo1 deletion background (Fig. 6). This finding provides a direct link between Glo1p and Met17p. This drop in Met17p-GFP expression could provide the basis for both phenotypes seen in the glo1 mutant: reduced growth on sulfate and accumulation of H2S. Reduced growth on sulfate is fully consistent with a previous finding that Met17p haploinsufficiency leads to impaired competition of this strain in synthetic complete medium;38 presumably a 50% reduction in expression of Met17p still provides sufficient homocysteine to allow slow growth on sulfate-containing media.

The H2S

accumulation phenotype of the glo1 mutant is also consistent with H2S production by the met17 null mutant (Fig 1.),10 and suggests that the reduced production of Met17p in this



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mutant provides insufficient enzyme activity to mop up all of the H2S produced by the SAP during the particular fermentation conditions used here. However, the Glo1p-overexpressing strain produced no significant differences in Met17pGFP compared to the wild type background (p > 0.05), although there appeared to be increased variability of Met17p-GFP expression in this strain background. This result is therefore not consistent with the phenotypes of the overexpressing strain, increased production of H2S and slow growth on sulfate; however, the variability in expression that was observed is consistent with a change in protein regulation, and it may be that analysis of additional time points of the fermentation profile would reveal differences. The difference could also be explained if GLO1 is not only regulating the expression of Met17p, but also that of another protein essential for the correct assembly of the O-acetylhomoserine sulfhydrylase protein complex. Glo1p has never been described as a transcriptional regulator, and indeed it does not contain any known domain in its protein structure indicating such a role. However, our data suggest a regulatory effect of GLO1 on MET17, where the correct expression of the former is required for the correct expression of the latter. The mechanism of this regulation needs to be further investigated.

Conclusion

In a venture to uncover yeast genes involved in the production of H2S during wine fermentation, we identified a novel gene function for glyoxylase I, encoded by GLO1. Yeast strains lacking GLO1 accumulate H2S, grow slowly on sulfate, and show reduced expression


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of Met17p. These effects occur independently of the levels of methylglyoxal to which to cells were exposed, and were not shown by the GLO2 or GLO4 genes downstream in the pathway. The discovery of this novel function for GLO1 in the well characterised sulfate assimilation pathway was surprising, and suggests that additional genes may yet be discovered that participate in, or interact with, this pathway. The GLO1 gene may provide an extra tool in the process of selecting an appropriate wine yeast strain that prevents the formation of H2S during grape juice fermentation, as well as furthering our understanding on the intermetabolic pathway complexities involved in the formation of signalling molecules.

Methods

Yeast strains and culture

This work primarily used single deletion mutants from the Saccharomyces Genome Deletion Project in the diploid BY4743 background. The major strains used in this study are listed in Table 1. Additional knockout strains in BY4743 that were tested included deletions of the following genes: MET10 (YFR030W), GRE2 (YOL151W), SNO4 (YMR322C), HSP31 (YDR533C), ADH1 (YOL086C), and GRE3 (YHR104W). The identity of the glo1 deletion strain was confirmed by testing for methylglyoxal sensitivity, and by amplification of kanamycin resistance from the strain using GLO1-specific oligos. YPD media (10 g/L yeast extract, 20 g/L peptone and 20 g/L glucose) was used for standard yeast culture at 28°C. Geneticin (G418; 200 mg/L) was added to the media for selecting strains with KanMX markers. Synthetic grape media (SGM) ferments.


22

was used for 100-mL


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Yeast growth rates were monitored using the Bioscreen C MBR plate incubator/reader/shaker controlled by the EZExperiment software (Oy Growth Curves AB Ltd, Helsinki, Finland). Yeast pre-cultures of ∼ 1.5×105 cells were added into a 100-well honeycomb plate containing 150 µL medium and grown for 72 h at 28°C in at least triplicates. The turbidity was measured at OD480–560 nm every 15 min with 75 s shaking prior to measurement.

Genetic manipulation and strain construction

The plasmid pE24Glo1, overexpressing GLO1,

15

was kindly provided by Prof Yoshiharu

Inoue, at Kyoto University, Japan. It was transformed into yeast by the high efficiency lithium acetate transformation method.39

Fermentation and H2S quantification

Fermentations were carried out in triplicate in 250-mL conical flasks containing 100 mL of SGM. Flasks were sealed with rubber stoppers fitted with H2S-detecting, silver nitrate tubes (Komyo Kitagawa, Tokyo, Japan).40 Fermentation of the knockout strains was performed at 28 °C, with shaking at 100 rpm and was monitored by daily weighing. Ferments were considered finished when weight loss was ≤ 0.1 g per 24 h. The supernatant was separated from yeast cells and solids by centrifugation at 6,000 g for 10 min and was stored frozen at −20 °C. The concentrations of H2S were recorded by visual reading from the tubes daily.

Indicator plates for H2S production


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BiYNB media plates (2% agar) contained bismuth ammonium citrate (5 g/L) in supplemented minimal media (yeast nitrogen base 5 g/L, ammonium sulfate 1.7 g/L, leucine 0.03 g/L, histidine 0.03 g/L, methionine 0.01 g/L, uracil 0.02 g/L). Potassium tartrate (5 g/L), L-malic acid (3 g/L), and citric acid (0.2 g/L), were added to adjust pH to that of synthetic grape media. Glucose was added to 0.2%, 2%, or 20% final concentration. Indicator plates were incubated at 28°C for 48 h and photographed.

Pull down assay

A BY4741 Glo1p-TAP strain containing a HIS3 marker gene, was purchased from Dharmacon (GE Healthcare, USA), and a BY4742 Met17p-GFP fusion protein strain was produced in our lab using geneticin selection following the published procedure.41 The Met17p-GFP fusion was functionally active, based on its growth in SGM not accumulating H2S during fermentation (data not shown). Both haploids were crossed, and the resulting BY4743-like diploid strain containing both tagged genes was selected in minimal media containing G418 (200 µg/mL), leucine (0.03 g/L), uracil (0.02 g/L) and lacking histidine. This resulting strain was grown in 50 mL of SGM to around ∼2×107 cells/mL. The pull-down assay was performed following the specifications for the GFP-Trap®_A kit, obtained from ChromoTek (Munich, Germany). Both anti-GFP and anti-TAP antibodies were obtained from Abcam®. The dot blot protocol followed was that specified by abcam.com/technical.

Polyacrylamide gel and western blot



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Yeast cells grown in SGM to ∼2×107 cells/mL were lysed by adding an equal volume of lysis buffer (100 mM Tris, 2% SDS, 10 M urea, 5% β-ME, 0.002% bromophenol blue, pH 7.0) and heated to 90°C for 5 min. Cell lysates were loaded onto a native or sodium dodecyl sulfate (SDS) containing 12% polyacrylamide (PAGE) gel and electrophoresed with running buffer (50 mM 3-(Nmorpholino)propanesulfonic acid (MOPS), 50 mM Tris, 0.1% SDS, pH 7.7) at 140 V, room temperature. The running buffer for the native PAGE did not contain SDS. Proteins were then transferred onto 0.2 µm nitrocellulose membrane (GE) with transfer buffer (25 mM Bicine, 25 mM Tris, 10% methanol, pH 7.2) at 100 V, 4°C for 1 h. The transfer buffer for the native PAGE differed in that it lacked methanol. The membrane was then blocked with 5% BSA in TBS-T (25 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.4) for at least 1 hour with gentle shaking. The membrane was probed with rabbit anti-GFP (Abcam ab290) at 1:2000 in 5% BSA in TBS-T for 1 h, washed at least 3 times in TBS-T and then probed with rabbit anti-GAPDH (Abcam ab9485) at 1:1000 in 5% BSA in TBS-T for at least 2 h with gentle shaking. After another cycle of washing, the membrane was finally probed with a secondary anti-rabbit antibody (Licor, IRDye 800CW) at 1:20000 for 1 h in the dark. Signal detection was carried out with an Odyssey® CLx Imaging System (Licor) and analysed with Image Studio. Band detection and signal were determined using setting: median with border 3. R studio was used to calculate relative expression levels, generate graphs and determine the levels of statistical significance with ANOVA, followed by Tukey Honest Significant Differences.

Acknowledgements


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We thank the reviewers of earlier versions of this manuscript whose suggestions for additional experiments have greatly helped strengthen the manuscript. We are grateful to Prof. Y. Inoue of Kyoto University for kindly supplying the pE24GLO1 overexpression plasmid. Special thanks to K. Ly, for his assistance in the use of the imaging techniques used in the dot blot, and to K. Richards and M. Huang for advice with the project. Funding for this project was provided by grant NZW 13-102 from New Zealand Winegrowers and the Romeo Bragato Trust.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:



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References:

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Sulfurilated amino acids 2-

SO

4

GLO1

MET17

Signaling

H2S GLO1 GLO1 ACS Paragon Plus Environment

GLO1

GLO1


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Fig. 1. A. The sulfate assimilation pathway (SAP) in yeast. B. The methylglyoxal degradation pathway showing GLO1 and GLO2.


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Fig. 2. Hydrogen sulfide production increases with added sulfate in both met17 and glo1 deletion strains. A: Time course of production of H 2 S (ppm) in 20-mL ferments. Colour code: glo1 deletion strain fermented in SGM (circle), met17 mutant in SGM (triangle), and glo1 mutant in SGM with sulfate (diamond), methionine (cross), cysteine (square) or GSH (full triangle) as its only sulfur source. B: Response of H 2 S production to added sulfate in 100-mL SGM cultures containing low methionine (0.075 mM). Lines represent wild-type BY4743 strain (purple), glo11 (red) and met17 (blue) in 0 mM (cross), 0.037 mM (star), 0.075 mM (circle), 0.15 mM (diamond), 0.5 mM (square) and 5 mM (triangle) final concentration sulfate. Bars show SD (n=3).



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Fig. 3. BiYNB plates with different glucose concentrations. Of the six glyoxylases tested (GLO1, GRE2, SNO4, HSP31, ADH1, GRE3 – see text), only the glo1 deletion presents H 2 S production. Deletions in met17 and met2 were used as positive controls. BY4743 (WT) and the met10 deletion were used as negative controls. H 2 S production is indicated on minimal YNB plates containing bismuth (BiYNB) by dark staining of the colonies as a result of bismuth sulfide formation. Left: 0.2%; Middle: 2%, Right: 20% glucose, respectively.


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Fig. 4. The glo1 deletion strain grows poorly on low sulfate, but not on methionine or cysteine. Bioscreen growth curves are shown with low concentrations of: A: Cysteine, B: Sulfate, C: Methionine. Colour code: Wild type BY4743 (blue), deletion strains in glo1 (red), met2 (purple, omitted in B), and met17 (green, omitted in A).



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Fig. 5. A Glo1p overexpression strain mimics the phenotype of the glo1 deletion strain, but no protein interactions were detected between Glo1p and Met17p via a pull-down assay. A: Bioscreen growth curves in SGM with 0.150 mM sulfate content. Colour code: Wild type BY4743 (blue), BY4743 + pE24GLO1 (red), glo1 (green), glo1 + pE24GLO1 (purple), and met17 (light blue). B: Colony colour on BiYNB plates (2% sugar) with strains overexpressing Glo1p. As in Fig. 2, H 2 S production is indicated by dark staining of the colonies as a result of bismuth sulfide formation.


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Fig. 6. Western blot quantification of Met17p. Above: Equal amounts of the various strains of yeast lysates were loaded in each lane and probed with anti-GFP and anti-GAPDH (load control) antibodies. First lane: BY4742 (wild type), second lane: BY4742 Met17p-GFP, third lane: BY4742 Met17p-GFP with glo1 deletion, and fourth lane: BY4742 Met17p-GFP with Glo1p overexpression. Below: Relative expression of Met17p-GFP. WT: BY4742 Met17pGFP, Del: Met17p-GFP with glo1 deletion, and Ox: BY4742 Met17p-GFP with Glo1p overexpression. Error bar: standard deviation (n=4). * p < 0.05.



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Table 1. Hydrogen sulfide detection after 24 h of fermentation H2S Strain

Genotype

Origin

production (ppm)

MATa/α his3Δ1/his3Δ1, leu2Δ0/leu2Δ0, LYS2/lys2Δ0, EUROSCARF collection BY4743

ND met15Δ0/MET15, ura3Δ0/ura3Δ0

(Frankfurt, Germany) EUROSCARF collection

BY4743 (YLR303W)

BY4743 with met17::KanMX/met17::KanMX

>1000 (Frankfurt, Germany) EUROSCARF collection

BY4743 (YNL277W)

BY4743 with met2::KanMX/met2::KanMX

>1000 (Frankfurt, Germany) EUROSCARF collection

BY4743 (YML004C)

>1000

BY4743 with glo1::KanMX/glo1::KanMX (Frankfurt, Germany) EUROSCARF collection

BY4743 (YDR272W)

BY4743 with glo2::KanMX/glo2::KanMX

ND (Frankfurt, Germany) EUROSCARF collection

BY4743 (YOR040W)

BY4743 with glo4::KanMX/glo4::KanMX

ND (Frankfurt, Germany)

ND, not detected. The hydrogen sulfide detection columns had a 1000 ppm limit that was reached within 24 h of fermentation in 100 mL of synthetic grape media


The GLO1 Gene Is Required for Full Activity of O ... - ACS Publications

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