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Article

GSM1 Requires Hap4 for Expression and Plays a Role in Gluconeogenesis and Utilization of Nonfermentable Carbon Sources

by
Manika Bhondeley
1,2 and
Zhengchang Liu
1,*
1
Department of Biological Sciences, University of New Orleans, New Orleans, LA 70148, USA
2
Kudo Biotechnology, 117 Kendrick Street, Needham, MA 02494, USA
*
Author to whom correspondence should be addressed.
Genes 2024, 15(9), 1128; https://doi.org/10.3390/genes15091128
Submission received: 31 July 2024 / Revised: 22 August 2024 / Accepted: 23 August 2024 / Published: 27 August 2024
(This article belongs to the Section Molecular Genetics and Genomics)
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Abstract

:
Multiple transcription factors in the budding yeast Saccharomyces cerevisiae are required for the switch from fermentative growth to respiratory growth. The Hap2/3/4/5 complex is a transcriptional activator that binds to CCAAT sequence elements in the promoters of many genes involved in the tricarboxylic acid cycle and oxidative phosphorylation and activates gene expression. Adr1 and Cat8 are required to activate the expression of genes involved in the glyoxylate cycle, gluconeogenesis, and utilization of nonfermentable carbon sources. Here, we characterize the regulation and function of the zinc cluster transcription factor Gsm1 using Western blotting and lacZ reporter-gene analysis. GSM1 is subject to glucose repression, and it requires a CCAAT sequence element for Hap2/3/4/5-dependent expression under glucose-derepression conditions. Genome-wide CHIP analyses revealed many potential targets. We analyzed 29 of them and found that FBP1, LPX1, PCK1, SFC1, and YAT1 require both Gsm1 and Hap4 for optimal expression. FBP1, PCK1, SFC1, and YAT1 play important roles in gluconeogenesis and utilization of two-carbon compounds, and they are known to be regulated by Cat8. GSM1 overexpression in cat8Δ mutant cells increases the expression of these target genes and suppresses growth defects in cat8Δ mutants on lactate medium. We propose that Gsm1 and Cat8 have shared functions in gluconeogenesis and utilization of nonfermentable carbon sources and that Cat8 is the primary regulator.

1. Introduction

The budding yeast S. cerevisiae can utilize different types of carbon sources to produce energy. In the presence of abundant glucose, yeast cells prefer to use fermentation to produce ATP, even in the presence of oxygen and non-fermentable carbon sources. During the diauxic shift or when the glucose level is low, there is a switch to respiratory metabolism for the generation of ATP, which requires a coordinated change in gene expression via multiple transcriptional regulatory factors, as reviewed in [1,2]. This leads to increased expression of genes encoding enzymes and proteins involved in gluconeogenesis, the glyoxylate cycle, the tricarboxylic acid cycle, and oxidative phosphorylation.
The change in metabolic programming during the transition to glucose-limiting conditions is regulated by a variety of regulatory proteins, including the Hap2/3/4/5 complex, Snf1, Adr1, Cat8, Rds2, Ert1, and Sip4 [3,4,5,6,7,8,9,10,11,12]. Snf1 is a subunit of a heterotrimeric complex that is activated upon phosphorylation by upstream kinases and promotes the glucose-derepression pathway for the utilization of alternative carbon sources by regulating the phosphorylation state of Cat8, Sip4, and Adr1 [5,6,8,13,14,15]. Snf1 is also involved in transcriptional control of Cat8 [5]. Adr1 is a zinc finger transcription factor that regulates genes for the utilization of lactate, glycerol, and ethanol [3,4]. Cat8, Sip4, Rds2, and Ert1 are zinc cluster transcription factors. Cat8 and Sip4 are subject to glucose repression, and SIP4 expression requires Cat8 [6,7,15]. Cat8 and Sip4 share common target genes, including those involved in the glyoxylate cycle and gluconeogenesis [6,16]. The targets of Rds2 include genes involved in gluconeogenesis, the tricarboxylic acid cycle, and the glyoxylate cycle [11]. The target genes of Ert1, as identified by chromatin immunoprecipitation assays, overlap with those of Adr1, Cat8, and Rds2 [10]. Genome-wide location analysis and transcriptome analysis reveal important overlaps among the targets of the transcriptional regulators Adr1, Cat8, Ert1, and Rds2 [4,11,12,17], indicating that yeast utilization of non-fermentable carbon sources requires an intricately regulated network of factors and their target genes.
Utilization of non-fermentable carbon sources requires ATP production via oxidative phosphorylation in the mitochondria. The Hap2/3/4/5 complex is a master regulator of mitochondrial biogenesis and energy metabolism in yeast [18,19,20]. Hap2/3/4/5 is a heterotetrameric complex that is activated by heme and regulates the transcription of genes involved in the tricarboxylic acid cycle and oxidative phosphorylation [21,22,23,24,25,26,27]. Hap4 is the regulatory subunit of this complex, and its expression is subject to glucose repression. The Hap2/3/5 trimer binds to CCAAT sequence elements in target gene promoters and requires Hap4 for transcriptional activation [28].
Gsm1, a zinc cluster transcription factor, has been proposed to regulate the expression of genes involved in gluconeogenesis (PCK1 and FBP1, W.G. Bao & M. Bolotin-Fukuhara, personal communication to the authors of the review paper) [1]. Genome-wide transcriptome analysis has shown that the expression of GSM1/YJL103C is similar to that of genes involved in cellular respiratory metabolism and that GSM1/YJL103C requires Hap2 for expression under glucose-derepression conditions [18,29]. Several chromatin immunoprecipitation (CHIP) analyses have identified potential Gsm1 targets [30,31,32], but none has been validated using gene or protein expression assays. A recent report shows that Rds2 and Gsm1 have overlapping and distinct targets [30]. The authors reported that gsm1Δ had no growth defects on non-fermentable carbon sources by itself or in combination with mutations in other genes involved in gluconeogenesis. In this report, we characterized the regulation of GSM1 and dissected its functions. We found Hap4 is essential for GSM1 expression under glucose-derepression conditions. We validated a number of Gsm1 targets and uncovered combinatorial regulation of gene expression by Gsm1 and Cat8. We demonstrate that Gsm1 has functions overlapping with those of Cat8 and that it is required for cell growth on lactate medium in cat8Δ mutant cells.

2. Materials and Methods

2.1. Growth Media, Growth Conditions, Strains, and Plasmids

Yeast strains were grown at 30 °C in YPD (1% Bacto Yeast Extract (Fisher Scientific, Waltham, MA, USA), 2% Bacto Peptone (Fisher Scientific), 2% glucose (Fisher Scientific)), YPL (1% Bacto Yeast Extract, 2% Bacto Peptone, 3.7% DL-Lactic acid (85%) (Sigma-Aldrich, St. Louis, MO, USA), adjusted to pH 5.3 using NaOH), YNBcasD (0.67% yeast nitrogen base (Fisher Scientific), 1% casamino acids (Fisher Scientific), 2% dextrose), YNBcas5D (similar to YNBcasD, with 5% dextrose), YNBcasR (0.67% yeast nitrogen base, 1% casamino acids, 2% raffinose (USBiological, Salem, MA, USA)), and a complete supplement mixture medium with raffinose as the carbon source (CSM-Raffinose) (0.67% yeast nitrogen base, 2% glucose, 0.6 g/L CSM minus uracil and leucine, 2% raffinose), as indicated in the text or in the figure legends. Amino acids and uracil were added to the growth medium at standard concentrations to cover auxotrophic requirements if required [33]. Agar (USBiological) was added at a final concentration of 2% for the solid medium. Cells were grown in liquid medium in a shaking incubator at 220 rpm and at 30 °C. Cells streaked on plate medium were incubated at 30 °C. The yeast strains and plasmids used in this study are listed in Table 1 and Table 2. Deletion mutant strains were constructed by transforming yeast with the required gene-knockout cassettes, dissecting sporulated heterozygous diploid strains, and/or crossing two mutant strains to obtain diploids for sporulation and dissection. Gene-deletion mutations were confirmed by PCR genotyping.

2.2. Yeast Transformation and β-Galactosidase Activity Assays

Yeast cells were freshly grown in YPD liquid medium and transformed using the high-efficiency method [38]. The YNBcasD medium, the SD medium supplemented with appropriate amino acids and uracil, and the YPD medium supplemented with geneticin were used to select yeast transformants based on the URA3, HIS3, and kanMX4 selection markers, respectively. For the β-galactosidase activity assays, the yeast strains were grown in growth medium as indicated in the text or in the figure legend at 30 °C for at least six generations to allow them to reach an OD600 of about 0.6 before collection. The cells were collected by centrifugation, and the β-galactosidase activity assays were conducted using o-nitrophenyl β-D-galactopyranoside (ONPG) as substrate, as described [33]. Two to six independent cultures were grown, and assays were carried out in duplicate for each sample. The data are presented as the mean ± standard deviation. The means of the β-galactosidase activity assay results were compared using a t-test. A “∗” in figures indicates a significant difference between the means of two groups of data (p < 0.05).

2.3. Serial Dilution of Cells for Growth Analysis

The wild-type and isogenic mutant strains were freshly grown on the YPD solid medium at 30 °C for 2–3 days. The cells were picked from a plate into sterile water and diluted to the same starting OD600 of 0.1. Five-fold serial dilutions were made using sterile 96-well plates and 8-channel pipettes. Then, 5 μL aliquots of serially diluted cell resuspensions were spotted on solid YPD and YPL solid media. The cells were grown for 2 to 4 days at 30 °C before pictures were taken for cell-growth analysis.

2.4. Cellular Extract Preparation, Immunoblotting, and Immunoprecipitation

The yeast strains were grown in growth medium as indicated at 30 °C for at least six generations to allow them to reach an OD600 of about 0.6, and total cellular proteins were prepared as described [39]. Briefly, 1 mL cell culture was mixed with 160 μL freshly prepared solution of 7.5% β-mercaptoethanol and 1.85N NaOH and incubated on ice for 10 min. Then, 84 μL 100% trichloroacetic acid (w/v) was then added, and the mixture was incubated on ice for 10 min before protein pellets were obtained by centrifugation at 21,000 g for 5 min. Protein samples were resuspended in SDS-PAGE sample buffer with 100 mM dithiothreitol and boiled for 3 min before being separated by SDS-polyacrylamide gel (7.5%) electrophoresis. The lanes were loaded with equivalent amounts of proteins based on the OD600 reading of the cell cultures. Pre-stained protein ladder (P7710S, New England Biolabs, Ipswich, MA, USA) was used in all protein gels. Proteins were transferred to the nitrocellulose membrane for immunoblotting. The following antibodies were used in this study: anti-GFP antibody B-2 (1:1000, Santa Cruz Biotechnology Inc., Dallas, TX, USA); anti-Pgk1 antibody (1:2000), rabbit polyclonal antibodies against recombinant yeast phosphoglycerate kinase generated by the lab; and HRP-conjugated goat anti-mouse secondary antibody (1:3000, catalog # 115-035-003, Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Chemiluminescence images of Western blots were captured using the Bio-Rad ChemiDoc MP imaging system (Hercules, CA, USA) and processed using Bio-Rad Image Lab software (version 6.1).

3. Results

3.1. GSM1 Expression Is Subject to Glucose Repression and Requires Hap4 under Glucose-Derepression Conditions

Large-scale gene expression profiling studies have shown that GSM1 expression increases during the diauxic shift and during growth on glycerol or ethanol [29,40]. To confirm that GSM1 expression is carbon source-dependent, we generated a GSM1−lacZ reporter gene by fusing a 924-bp promoter of GSM1 to the bacterial lacZ gene. The plasmid encoding the GSM1−lacZ reporter gene was transformed into a wild-type BY4741 strain, and transformants were grown in media with dextrose or raffinose as the sole carbon source. Raffinose is a trisaccharide and a glucose-derepression carbon source. In the wild-type strain, a β-galactosidase activity assay shows that the expression of GSM1-lacZ is very low in glucose-grown cells, at a level close to the detection limit of the β-galactosidase activity assay using ONPG as the substrate. There is 5.2-fold higher expression in raffinose-grown cells compared to dextrose-grown cells (Figure 1A), a finding consistent with published microarray data.
Many genes require the Hap2/3/4/5 complex for their expression in cells grown under glucose-derepression conditions [18]. In the same study, a hap2∆ mutation was reported to reduce the expression of GSM1. To determine whether GSM1 is a target of Hap4, we introduced the plasmid encoding the GSM1-lacZ reporter gene into hap4∆ mutant cells and β-galactosidase activity assays were carried out. Figure 1A shows that a hap4∆ mutation reduces GSM1-lacZ expression by 45% in glucose-grown cells and by 7.3-fold in raffinose-grown cells, indicating that GSM1 expression is under the control of Hap4. In wild-type cells, there was a 5.2-fold increase in GSM1-lacZ activity in raffinose-grown cells compared to glucose-grown cells (Figure 1A). This increase in GSM1 expression was completely blocked in hap4∆ mutant cells. Together, our data suggest that GSM1 is a new target of Hap4.
Our data in Figure 1A clearly indicates that hap4Δ reduces GSM1 expression in cells grown in raffinose medium. A change in gene transcription does not always lead to a corresponding change in the protein level. Therefore, to determine whether the transcriptional control of GSM1 correlates with the protein level of Gsm1, we generated a plasmid encoding Gsm1 with a C-terminal GFP tag and introduced it into gsm1∆ cells. Transformants were grown in dextrose and raffinose medium. As a control, gsm1∆ cells carrying an empty vector were cultured similarly. The expression of the Gsm1-GFP fusion protein was detected by Western blotting with an anti-GFP antibody. gsm1∆ cells carrying the plasmid encoding the Gsm1-GFP fusion protein cultured in raffinose medium, but not those cultured in glucose medium, yielded a single band of ~100 kD, close to the predicted size of 98.7 kD (Figure 1B). Similar results were also obtained from an otherwise wild-type strain expressing GSM1-GFP (Figure 1C). In contrast, hap4Δ mutant cells expressing GSM1-GFP grown in raffinose medium did not yield a visible signal for Gsm1-GFP. Together, our data indicate that expression of GSM1 under glucose-derepression conditions requires Hap4.

3.2. A Proximal CCAAT Sequence Element Is Required for GSM1 Expression

The Hap2/3/4/5 complex activates the expression of its target genes by binding to CCAAT sequence elements in their promoter region under glucose-derepression conditions, as reviewed in [41]. The promoter region of GSM1 contains two CCAAT sequence elements at −634 bp and at −286 bp in relation to the ATG start codon (Figure 2A). To determine which CCAAT element confers Hap4-dependent GSM1 expression, we mutated each of the CCAAT sequences into the TCACA sequence and generated a site 1 mutation (S1M) at the −634 bp position and a site 2 mutation (S2M) at the −286 bp position (Figure 2A). The effect of the two mutations in the reporter constructs GSM1(S1M)-lacZ and GSM1(S2M)-lacZ was examined in wild-type BY4741 cells grown in dextrose and raffinose media. In both dextrose- and raffinose-grown cells, the CCAAT-element mutation at the −634 bp position (GSM1(S1M)-lacZ) did not lead to significant changes in the expression of the lacZ reporter gene. In contrast, the mutation in the CCAAT sequence element at the −286 bp position reduced GSM1-lacZ expression by 39% in glucose-grown cells and by 7.4-fold in raffinose-grown cells. The almost identical effects on GSM1-lacZ expression caused by a hap4∆ mutation and a mutation in the proximal CCAAT sequence element (compare Figure 1A with Figure 2B) indicate that Hap4 directly regulates the expression of GSM1 via the CCAAT sequence at the −286 bp position.

3.3. Identification of Potential Gsm1 Target Genes via Analysis of lacZ Reporter Genes

A genome-wide study of Gsm1 binding sites revealed potential Gsm1 target genes via a chromatin immunoprecipitation (CHIP) analysis [31]. The authors introduced a new method that involved two rounds of T7 RNA polymerase amplification (double-T7) to amplify ChIP DNA for microarray analysis. The double-T7 method showed stronger binding signals compared to traditional ligation-mediated polymerase chain reaction (LM-PCR). Figure 3A shows an example, FBP1, as a potential target of Gsm1 that was identified using these two methods. We used the data from this study to select target genes and chose 29 potential targets (ADR1, ERG3, FBP1, GAT2, GID7, GID8, HAP4, IDP2, LPX1, MDH2, MDY2, MIR1, MOT3, MSN2, MSN4, PCK1, PRB1, PYC1, PYK2, RCF2, RHO5, ROX1, SFC1, SHR5, SIT4, TSL1, VHR1, YAT1, and ZWF1) based on the binding signals from both the T7-based amplification method and conventional LM-PCR. Two of the 29 genes, ADR1 and HAP4, were also identified in another Gsm1 CHIP assay [32]. While we were preparing the manuscript, another report on the Gsm1 targets was published; this report included PCK1, HAP4, and FBP1 as potential targets [30]. In all three reports on Gsm1 targets identified via CHIP analysis, the target genes were not confirmed by gene- or protein-expression analysis. We generated 29 lacZ reporter genes, each of which was under the control of a different gene promoter listed above. Plasmids bearing lacZ reporter genes were transformed into wild-type and gsm1∆ cells. Transformants were grown in raffinose medium and β-galactosidase activity assays were conducted. Figure 3B shows that the expression of 10 reporter genes, namely, FBP1, IDP2, LPX1, MDY2, MSN4, PYC1, PYK2, YAT1, ZWF1, and GAT2, was significantly reduced (p < 0.05) in gsm1∆ mutant cells. Five out of these 29 genes, GID7, MDH2, PCK1, SFC1, and SHR5, show reduced expression, with a p value close to the 0.05 cut-off, in gsm1∆ mutant cells compared to wild-type cells. Most of the lacZ reporter genes show reduced expression in gsm1∆ mutant cells, a finding consistent with the notion that Gsm1 is a transcriptional activator. Only two genes, VHR1 and HAP4, show an increase in expression of more than 5% in gsm1∆ cells.
Among the 15 genes showing a reduction in their expression in gsm1∆ cells with a p value less than or close to 0.05, nine of them, FBP1, IDP2, LPX1, PCK1, PYC1, PYK2, SFC1, YAT1, and ZWF1, are involved in gluconeogenesis, carbohydrate metabolism, and/or the utilization of non-fermentable carbon sources. We chose five for further analysis: FBP1 (encoding fructose-1,6-bisphosphatase), PCK1 (encoding phosphoenolpyruvate carboxykinase), SFC1 (encoding a mitochondrial succinate-fumarate transporter), LPX1 (encoding a peroxisomal matrix-localized lipase), and YAT1 (encoding outer mitochondrial carnitine acetyltransferase). Figure 3C shows that expression of FBP1-, PCK1-, LPX1-, SFC1-, and YAT1-lacZ reporter gene are reduced in both gsm1Δ and hap4Δ mutant cells grown in the raffinose medium. Since Hap4 is strictly required for GSM1 expression in this medium (Figure 1), it is expected that hap4Δ reduces the expression of these five genes. In fact, compared to gsm1Δ, hap4Δ leads to a greater reduction in the expression of FBP1, LPX1, SFC1, and YAT1, suggesting that Hap4 may regulate other transcription factors, which in turn mediate the expression of these four genes.
We have shown that Hap4 is required for GSM1 expression under glucose-derepression conditions (Figure 1). The three reports on the CHIP analysis of Gsm1 all show HAP4 is a potential target [30,31,32]. However, we failed to see reduced expression of HAP4-lacZ in gsm1Δ mutant cells (Figure 3C). On the contrary, among the 29 genes we analyzed, HAP4-lacZ was the only reporter gene that showed a small induction, with a p value close to 0.05 (p = 0.16), in gsm1Δ cells (Figure 3B). In contrast, hap4Δ reduces HAP4 expression by 2.3-fold, a result consistent with published findings [27]. It appears that a strong binding based on CHIP data does not necessarily translate into transcriptional regulation. Rds2 has been reported to be required for HAP4 expression [11], but we failed to see decreased expression of HAP4-lacZ in rds2Δ mutant cells (BY4741 background) grown in raffinose. The discrepancy may be attributed to a difference in the growth conditions (ethanol versus raffinose).

3.4. GSM1 Overexpression Increases Expression of FBP1-, LPX1-, PCK1-, SFC1-, and YAT1-lacZ Reporter Genes in hap4∆ Mutant Cells

We have shown that Hap4 is strictly required for GSM1 expression in cells grown in the raffinose medium. Figure 3C shows that the effect of hap4Δ on reducing the expression of the FBP1-, LPX1-, PCK1-, SFC1-, and YAT1-lacZ reporter genes is stronger than that of gsm1Δ. We decided to test the effect of GSM1 overexpression on the expression of these five reporter genes in both wild-type and hap4Δ mutant cells. Accordingly, wild-type and hap4Δ mutant cells expressing the lacZ reporter genes were transformed with a plasmid encoding Gsm1 with a C-terminal 3× myc tag under the control of the strong TEF2 promoter (TEF2-GSM1-myc) or with the empty vector pRS415TEF, which served as a control. Transformants were selected on complete supplement mixture medium without uracil and leucine to select for and maintain the plasmid carrying the URA3 or LEU2 marker. Transformants were grown in CSM-Raffinose medium without uracil and leucine, and a β-galactosidase activity assay was conducted. Figure 4A shows that TEF2-GSM1-myc increases the expression of these five lacZ reporter genes in both wild-type and hap4Δ mutant cells, consistent with the notion that these five genes are Gsm1 targets. In all reporter genes except YAT1-lacZ, TEF2-GSM1-myc leads to a significantly higher expression level in wild-type than in hap4Δ mutant cells, suggesting that other Hap4-dependent proteins also contribute to the regulation of FBP1, LPX1, PCK1, and SFC1.
After establishing a functional essay for GSM1, we decided to determine whether the GSM1-GFP construct used to produce the results in Figure 1B,C was functional. gsm1Δ mutant cells expressing FBP1-lacZ were transformed with a plasmid encoding GSM1-GFP under the control of the GSM1 promoter or with an empty vector, which served as the control. Transformants were grown in CSM-raffinose medium, and a β-galactosidase activity assay was conducted. Figure 4B shows that GSM1-GFP largely complements gsm1Δ by restoring FBP1-lacZ expression. Since Gsm1-GFP was largely functional, we can conclude that the protein-expression result presented in Figure 1B,C is physiologically relevant.

3.5. Cat8 and Gsm1 Are Important in the Transcriptional Regulation of FBP1, PCK1, SFC1, and YAT1 and in the Utilization of Lactate

Yeast cells undergo a transcriptional switch in order to utilize nonfermentable carbon sources. There is a dramatic upregulation of genes involved in gluconeogenesis, the glyoxylate cycle, the tricarboxylic acid cycle, and oxidative phosphorylation. Genome-wide transcriptome analyses have shown that Cat8 and Adr1 are required for derepression of many genes important for utilization of nonfermentable carbon sources [4,16,17]. Among the five genes we chose for further analysis as Gsm1 targets, FBP1, PCK1, SFC1, and YAT1 are known to be regulated by Cat8 [16]. To analyze whether FBP1, LPX1, PCK1, SFC1, and YAT1 are subject to combinatorial regulation by Gsm1, Adr1, and Cat8, we generated all possible double and triple mutants from cat8∆, gsm1∆, and adr1∆ mutations. We transformed FBP1-lacZ, LPX1-lacZ, PCK1-lacZ, SFC1-lacZ, and YAT1-lacZ reporter constructs into adr1∆, cat8∆, gsm1∆, adr1∆ cat8∆, adr1∆ gsm1∆, cat8∆ gsm1∆, and adr1∆ cat8∆ gsm1∆ mutant cells. Transformants were grown in YNBcasR medium, and a β-galactosidase activity assay was carried out. Figure 5A,C,E shows that there is combinatorial regulation of the FBP1-, PCK1-, and YAT1-lacZ reporter genes by Cat8 and Gsm1, since the expression levels of these three lacZ reporter genes in the cat8∆ gsm1∆ double mutant are lower than those observed in the cat8∆ and gsm1∆ single mutants. Although SFC1-lacZ is significantly reduced in gsm1∆ mutant cells, it is difficult to determine whether SFC1 is subject to combinatorial regulation by Cat8 and Gsm1 due to the extremely low expression level already present in the cat8Δ single mutant (Figure 5D).
An adr1Δ mutation does not result in significant changes in the expression of the FBP1-, PCK1-, SFC1-, and YAT1-lacZ reporter genes (Figure 5A,C–E), which is consistent with published findings [4]. Consistently, while Cat8 and Gsm1 are required for the expression of these four reporter genes, any additional combinatorial regulation by Adr1 is either minimal or nonexistent. Together, our data suggest that Cat8 and Gsm1 have related functions and that Cat8 is the primary regulator of the two.
Adr1 and Cat8 play important roles in the regulation of genes involved in the utilization of nonfermentable carbon sources. Our finding that Cat8 and Gsm1 have related functions prompted us to test the growth phenotypes of adr1∆, cat8∆, gsm1∆, adr1∆ cat8∆, adr1∆ gsm1∆, cat8∆ gsm1∆, and adr1∆ cat8∆ gsm1∆ mutant cells on growth medium with dextrose or lactate as the sole carbon source (Figure 5F). On the dextrose medium, none of the mutants exhibited growth defects, which is consistent with the role of these transcription factors in the utilization of nonfermentable carbon sources. On the lactate medium, cat8Δ led to significant growth defects while adr1∆, gsm1∆, adr1∆ gsm1∆ mutant cells had the same level of growth as wild-type cells. Importantly, while gsm1∆ did not lead to growth defects, the residual growth in cat8Δ mutant cells was eliminated in the cat8∆ gsm1∆ double- and adr1∆ cat8∆ gsm1∆ triple mutant cells. Together, our data suggest that Cat8 and Gsm1 have related functions and that Cat8 is the primary regulator.

3.6. GSM1 Overexpression Increases Target-Gene Expression in adr1∆ cat8∆ Mutant Cells and Suppresses Growth Defects Associated with cat8Δ

Our data, as represented in Figure 5, show that Cat8 and Gsm1 have related functions in the regulation of genes involved in the utilization of nonfermentable carbon sources. Adr1 is also important for this function. We decided to test whether GSM1 overexpression can increase the expression of the target genes in the absence of both Adr1 and Cat8. Accordingly, wild-type and adr1∆ cat8∆ mutant cells expressing FBP1-lacZ, LPX1-lacZ, PCK1-lacZ, SFC1-lacZ, and YAT1-lacZ reporter genes were transformed with a plasmid encoding GSM1 with a 3x myc tag at the C-terminus under the control of the strong TEF2 promoter. Transformants were grown in CSM-raffinose medium, and a β-galactosidase assay was conducted. Figure 6A shows that GSM1 overexpression in adr1∆ cat8∆ mutant cells significantly increased the expression of all five reporter genes, indicating that Gsm1 can activate the expression of its target genes independent of Adr1 and Cat8. Figure 6A also shows that GSM1 overexpression in wild-type cells led to higher levels of target gene expression than did its overexpression in adr1∆ cat8∆ mutant cells. This result suggests that, although Gsm1 can function independently of Adr1 and Cat8, it requires Adr1 and/or Cat8 for maximum expression of FBP1-lacZ, LPX1-lacZ, PCK1-lacZ, SFC1-lacZ, and YAT1-lacZ reporter genes.
Next, we wanted to determine whether GSM1 overexpression can suppress the severe growth defects of cat8∆ mutant cells on lactate medium. We overexpressed GSM1 under the control of the TEF2 promoter in adr1∆, cat8∆, and cat8∆ adr1∆ cells, which were serially diluted and spotted on YPD and YPL media. Figure 6B shows that GSM1 overexpression suppressed the growth defects of cat8∆ and cat8∆ adr1∆ double mutant cells on lactate medium. Together, our data indicate that Gsm1 and Cat8 have related functions via combinatorial regulation of target gene expression, which in turn enables cells to utilize non-fermentable carbon sources.

4. Discussion

In this report, we characterized the regulation and function of the zinc cluster transcription factor Gsm1. GSM1 is subject to glucose repression and requires Hap4 for its induced expression under glucose-derepression conditions. A number of targets genes based in CHIP data were validated, including FBP1, LPX1, PCK1, SFC1, and YAT1. We found that gsm1Δ and cat8Δ have additive effects on the reduction of target gene expression and that GSM1 overexpression suppresses the severe growth defects of cat8Δ mutant cells grown on a non-fermentable carbon source. We propose that Gsm1 and Cat8 have shared functions in regulating target gene expression and enabling yeast cells to utilize non-fermentable carbon sources.
GSM1 expression is subject to glucose repression (Figure 1), a mechanism similar to that regulating the expression of CAT8 and SIP4. This is achieved by Hap4-dependent activation under glucose-derepression conditions. The expression of HAP4, like that of its target genes, is also subject to glucose repression. In glucose-grown cells, low levels of HAP4 expression translate to reduced activity of the Hap2/3/4/5 complex, which leads to basal expression of GSM1. The strategy of maintaining low expression levels of GSM1, CAT8, and SIP4 is logical since they are not required in cells grown in glucose. The Hap2/3/4/5 complex binds to CCAAT sequence elements in the promoter of the target genes, leading to their transcriptional activation. The promoter of GSM1 contains two CCAAT sequence elements. We found that the one close to the ATG start codon is essential for GSM1 expression under glucose-derepression conditions, indicating that Hap2/3/4/5 regulation of GSM1 expression is direct. Due to the interdependent regulation of genes encoding transcription factors involved in the transition to nonfermentable growth, it will be a challenge to determine whether the regulation of a target gene expression by a transcriptional regulator is direct or indirect.
The function of Gsm1 has not been clear until this study. Three genome-wide CHIP analyses have revealed potential Gsm1 targets, but they have not been validated by gene- or protein-expression assays. Among 29 genes we analyzed, we found that 12 genes, FBP1, IDP2, LPX1, MDY2, MSN4, PCK1, PYC1, PYK2, SFC1, YAT1, ZWF1, and GAT2, are Gsm1 targets. As shown in Figure 3B, the p values for the differences in the expression of PCK1 and SFC1 in wild-type cells versus in a gsm1 mutant were 0.14 and 0.17, respectively. These values are not considered statistically different. However, the higher p values were due to the analysis of only two independent cultures of yeast strains expressing PCK1 or SFC1 while most strains carrying the reporter genes were represented by four independent cultures. As seen in Figure 4, Figure 5 and Figure 6, PCK1 and SFC1 are authentic targets of Gsm1. Among the 12 genes, FBP1, PCK1, and PYC1 encode enzymes in the gluconeogenesis pathway and SFC1 is important for the utilization of ethanol and acetate. Thus, Gsm1 is involved in gluconeogenesis and the utilization of nonfermentable carbon sources.
It has been proposed that Gsm1 is important for oxidative phosphorylation [40], a suggestion based on the analysis of genes having expression profiles similar to that of GSM1. Since GSM1 is under the control of Hap2/3/4/5 complex, its expression profile should be similar to that of genes involved in the tricarboxylic acid cycle and oxidative phosphorylation. We believe this conclusion is misleading. HAP4 has been highlighted as a potential GSM1 target [30,31,32]. We failed to detect reduced HAP4 expression in gsm1Δ mutant cells grown in raffinose medium (Figure 3C). On the contrary, a slight increase in the expression of HAP4 in gsm1Δ mutant cells was detected. This may be an example in which the binding of a transcription factor to the promoter does not lead to altered gene expression. It is possible that Gsm1 binding can lead to increased expression of HAP4 under other growth conditions.
Among the 12 genes we identified as Gsm1 targets, FBP1, IDP2, PCK1, SFC1, and YAT1 are also targets of Cat8 [16]. In this report, we show that cat8Δ and gsm1Δ have additive effects on target gene expression and that GSM1 overexpression can rescue reduced gene expression caused by cat8Δ (Figure 5 and Figure 6). We also found that gsm1Δ and cat8Δ have an additive effect in reducing cell growth on lactate medium and that GSM1 overexpression can suppress the severe growth defects caused by cat8Δ (Figure 5 and Figure 6). Taking these results together, we propose that Gsm1 and Cat8 have related functions in gluconeogenesis and the utilization of non-fermentable carbon sources and that Cat8 is the primary regulator of the two. Identification of Gsm1 as another regulator of genes involved in the utilization of nonfermentable carbon sources adds to the already complex network of transcriptional regulators involved in the process. Future work is needed to determine why yeast cells employ so many regulators with overlapping functions and cross-regulations to utilize carbon sources.

Author Contributions

M.B. conducted the experiments and data analysis, generated the figures, and wrote the first draft of the paper. Z.L. collaborated on the experiments, coordinated the study, edited the data figures and paper. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the NIH grant 1R15GM121998-01.

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Acknowledgments

We thank Natasha M. Bourgeois for technical support.

Conflicts of Interest

Author Manika Bhondeley was employed by the company Kudo Biotechnology. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. GSM1 expression is subject to glucose repression and requires Hap4 for induced expression under glucose-derepression conditions. (A) Wild-type (BY4741) and hap4∆ mutant cells (ZLY2811) carrying a plasmid encoding the GSM1-lacZ reporter gene (pZL3454) were grown to mid-logarithmic phase in YNBcas5D (Dextrose) and YNBcasR (Raffinose) medium. β-galactosidase activity assays were conducted as described in the Materials and Methods. The data are presented as the mean ± standard deviation. The means of the results were compared by a t-test. “∗” indicates a significant difference (p < 0.05) between the means of two groups of data indicated by the beginning and end of the horizontal line. (B,C) Wild-type (BY4741), hap4∆ (ZLY2811), and gsm1∆ (MBY123) mutant cells carrying a plasmid encoding GSM1-GFP (pZL3462) as indicated were grown in YNBcas5D (dextrose) and YNBcasR (raffinose) medium to mid-logarithmic phase, and total cellular proteins were prepared and probed by Western blotting using an anti-GFP antibody, as described in the Materials and Methods. Pgk1 was included as a loading control. The result was representative of three independent experiments for panel (B) and of two independent experiments for panel (C).
Figure 1. GSM1 expression is subject to glucose repression and requires Hap4 for induced expression under glucose-derepression conditions. (A) Wild-type (BY4741) and hap4∆ mutant cells (ZLY2811) carrying a plasmid encoding the GSM1-lacZ reporter gene (pZL3454) were grown to mid-logarithmic phase in YNBcas5D (Dextrose) and YNBcasR (Raffinose) medium. β-galactosidase activity assays were conducted as described in the Materials and Methods. The data are presented as the mean ± standard deviation. The means of the results were compared by a t-test. “∗” indicates a significant difference (p < 0.05) between the means of two groups of data indicated by the beginning and end of the horizontal line. (B,C) Wild-type (BY4741), hap4∆ (ZLY2811), and gsm1∆ (MBY123) mutant cells carrying a plasmid encoding GSM1-GFP (pZL3462) as indicated were grown in YNBcas5D (dextrose) and YNBcasR (raffinose) medium to mid-logarithmic phase, and total cellular proteins were prepared and probed by Western blotting using an anti-GFP antibody, as described in the Materials and Methods. Pgk1 was included as a loading control. The result was representative of three independent experiments for panel (B) and of two independent experiments for panel (C).
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Figure 2. A mutation in the CCAAT sequence at position −286 blocks increased expression of GSM1 under glucose-derepression conditions. (A) Diagrammatic representation of a 924 bp-long promoter sequence of GSM1 fused to the lacZ gene. The GSM1-lacZ reporter construct has two CCAAT sequence elements at positions −634 and −286 in relation to the ATG start codon. The mutations to the CCAAT sequences in the GSM1 promoter are indicated in red. (B) Wild-type cells (BY4741) carrying a plasmid encoding GSM1-lacZ (pZL3454), GSM1(S1M)-lacZ (pMB165), or GSM1(S2M)-lacZ (pMB168) were grown in YNBcas5D (dextrose) and YNBcasR (raffinose) medium, and β-galactosidase activity assays were conducted as described in the Materials and Methods. The data are presented as the mean ± standard deviation. ∗, p < 0.05.
Figure 2. A mutation in the CCAAT sequence at position −286 blocks increased expression of GSM1 under glucose-derepression conditions. (A) Diagrammatic representation of a 924 bp-long promoter sequence of GSM1 fused to the lacZ gene. The GSM1-lacZ reporter construct has two CCAAT sequence elements at positions −634 and −286 in relation to the ATG start codon. The mutations to the CCAAT sequences in the GSM1 promoter are indicated in red. (B) Wild-type cells (BY4741) carrying a plasmid encoding GSM1-lacZ (pZL3454), GSM1(S1M)-lacZ (pMB165), or GSM1(S2M)-lacZ (pMB168) were grown in YNBcas5D (dextrose) and YNBcasR (raffinose) medium, and β-galactosidase activity assays were conducted as described in the Materials and Methods. The data are presented as the mean ± standard deviation. ∗, p < 0.05.
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Figure 3. Transcriptional analysis of potential Gsm1 target genes using lacZ reporter gene analysis. (A) Gsm1-binding ratios of the FBP1 locus on the chromosome, as determined using double-T7 and LM-PCR methods [31]. (B) The expression ratios of 29 lacZ reporter genes in gsm1Δ mutant cells versus wild-type cells grown in YNBcasR medium. ∗, p < 0.05. The numbers on top of the bars are p values close to the 0.05 cut-off. The white bars indicate genes selected for further analysis. (C) A β-galactosidase activity assay on the expression of FBP1-, LPX1-, PCK1-, SFC1, YAT1-, and HAP4-lacZ reporter genes in wild-type (BY4741), hap4Δ (ZLY2811), and gsm1Δ (MBY123) mutant cells grown in YNBcasR medium. ∗, p < 0.05. FBP1-lacZ, pMB179; LPX1-lacZ, pMB181; PCK1-lacZ, pZL3628; SFC1-lacZ, pMB209; YAT1-lacZ, pZL3625; HAP4-lacZ, pDC124.
Figure 3. Transcriptional analysis of potential Gsm1 target genes using lacZ reporter gene analysis. (A) Gsm1-binding ratios of the FBP1 locus on the chromosome, as determined using double-T7 and LM-PCR methods [31]. (B) The expression ratios of 29 lacZ reporter genes in gsm1Δ mutant cells versus wild-type cells grown in YNBcasR medium. ∗, p < 0.05. The numbers on top of the bars are p values close to the 0.05 cut-off. The white bars indicate genes selected for further analysis. (C) A β-galactosidase activity assay on the expression of FBP1-, LPX1-, PCK1-, SFC1, YAT1-, and HAP4-lacZ reporter genes in wild-type (BY4741), hap4Δ (ZLY2811), and gsm1Δ (MBY123) mutant cells grown in YNBcasR medium. ∗, p < 0.05. FBP1-lacZ, pMB179; LPX1-lacZ, pMB181; PCK1-lacZ, pZL3628; SFC1-lacZ, pMB209; YAT1-lacZ, pZL3625; HAP4-lacZ, pDC124.
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Figure 4. (A) GSM1 overexpression increases the expression of its candidate target genes in hap4∆ mutant cells. Wild-type (BY4741) and hap4∆ mutant cells (ZLY2811) expressing a lacZ reporter gene as indicated were transformed with a centromeric plasmid overexpressing GSM1 under the control of the TEF2 promoter (TEF2-GSM1-myc, pZL3459) or with the empty vector (Vector, pRS415TEF), which served as a control. Transformants were grown to mid-logarithmic phase in a complete supplement mixture medium with raffinose as the carbon source (CSM-raffinose), and β-galactosidase activity assays were conducted. ∗, p < 0.05. (B) The GSM1-GFP construct was largely functional. Wild-type (BY4741) and gsm1∆ mutant cells (MBY123) carrying a plasmid encoding FBP1-lacZ and a plasmid encoding GSM1-GFP (pZL3613) or carrying the empty vector (Vector, pRS415) were grown to mid-logarithmic phase in CSM-raffinose medium, and β-galactosidase activity assays were conducted. ∗, p < 0.05.
Figure 4. (A) GSM1 overexpression increases the expression of its candidate target genes in hap4∆ mutant cells. Wild-type (BY4741) and hap4∆ mutant cells (ZLY2811) expressing a lacZ reporter gene as indicated were transformed with a centromeric plasmid overexpressing GSM1 under the control of the TEF2 promoter (TEF2-GSM1-myc, pZL3459) or with the empty vector (Vector, pRS415TEF), which served as a control. Transformants were grown to mid-logarithmic phase in a complete supplement mixture medium with raffinose as the carbon source (CSM-raffinose), and β-galactosidase activity assays were conducted. ∗, p < 0.05. (B) The GSM1-GFP construct was largely functional. Wild-type (BY4741) and gsm1∆ mutant cells (MBY123) carrying a plasmid encoding FBP1-lacZ and a plasmid encoding GSM1-GFP (pZL3613) or carrying the empty vector (Vector, pRS415) were grown to mid-logarithmic phase in CSM-raffinose medium, and β-galactosidase activity assays were conducted. ∗, p < 0.05.
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Figure 5. Cat8 and Gsm1 are important in the transcriptional regulation of FBP1, PCK1, SFC1, and YAT1 and in the utilization of lactate. (A–E) β-galactosidase activity assays of the expression of lacZ reporter genes, as indicated in wild-type (BY4741), adr1∆ (ZLY3707), cat8∆ (ZLY3701), gsm1∆ (MBY123), adr1∆ cat8∆ (ZLY5048), adr1∆ gsm1∆ (ZLY5103), cat8∆ gsm1∆ (ZLY5081), and adr1∆ gsm1∆ cat8∆ (ZLY5109) mutant cells grown in YNBcasR medium. The data are presented as the mean ± standard deviation. ∗, p < 0.05. (F) gsm1∆ exacerbates the growth defect of cat8∆ mutant cells grown on lactate medium. Yeast strains as described for panels (A–E) were serially diluted and spotted on YPD (dextrose) and YPL (lactate) medium. Pictures of the plates were taken after 2–4 days’ growth at 30 °C.
Figure 5. Cat8 and Gsm1 are important in the transcriptional regulation of FBP1, PCK1, SFC1, and YAT1 and in the utilization of lactate. (A–E) β-galactosidase activity assays of the expression of lacZ reporter genes, as indicated in wild-type (BY4741), adr1∆ (ZLY3707), cat8∆ (ZLY3701), gsm1∆ (MBY123), adr1∆ cat8∆ (ZLY5048), adr1∆ gsm1∆ (ZLY5103), cat8∆ gsm1∆ (ZLY5081), and adr1∆ gsm1∆ cat8∆ (ZLY5109) mutant cells grown in YNBcasR medium. The data are presented as the mean ± standard deviation. ∗, p < 0.05. (F) gsm1∆ exacerbates the growth defect of cat8∆ mutant cells grown on lactate medium. Yeast strains as described for panels (A–E) were serially diluted and spotted on YPD (dextrose) and YPL (lactate) medium. Pictures of the plates were taken after 2–4 days’ growth at 30 °C.
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Figure 6. (A) GSM1 overexpression increases the expression of its target genes in adr1∆ cat8∆ mutant cells. Wild-type (BY4741) and adr1∆ cat8∆ mutant cells (ZLY5048) expressing the lacZ reporter gene, as indicated, were transformed with a centromeric plasmid overexpressing GSM1 under the control of the TEF2 promoter (TEF2-GSM1-myc, pZL3459) or with an empty vector (Vector, pRS415TEF) as the control. Transformants were grown to mid-logarithmic phase in complete supplement mixture medium (CSM) with raffinose as the carbon source and β-galactosidase activity assays were conducted. ∗, p < 0.05. (B) GSM1 overexpression suppresses the growth defects of cat8∆ single and adr1∆ cat8∆ double mutant cells on lactate medium. Wild-type (BY4741), adr1∆ (ZLY3707), cat8∆ (ZLY3701), and adr1∆ cat8∆ (ZLY5048) mutant cells carrying a plasmid encoding TEF2-GSM1-myc (pZL3459) or carrying the empty plasmid (Vector, pRS415TEF) were serially diluted and spotted on YPD (dextrose) and YPL (lactate) medium. Pictures of the plates were taken after 2–4 days’ growth at 30 °C.
Figure 6. (A) GSM1 overexpression increases the expression of its target genes in adr1∆ cat8∆ mutant cells. Wild-type (BY4741) and adr1∆ cat8∆ mutant cells (ZLY5048) expressing the lacZ reporter gene, as indicated, were transformed with a centromeric plasmid overexpressing GSM1 under the control of the TEF2 promoter (TEF2-GSM1-myc, pZL3459) or with an empty vector (Vector, pRS415TEF) as the control. Transformants were grown to mid-logarithmic phase in complete supplement mixture medium (CSM) with raffinose as the carbon source and β-galactosidase activity assays were conducted. ∗, p < 0.05. (B) GSM1 overexpression suppresses the growth defects of cat8∆ single and adr1∆ cat8∆ double mutant cells on lactate medium. Wild-type (BY4741), adr1∆ (ZLY3707), cat8∆ (ZLY3701), and adr1∆ cat8∆ (ZLY5048) mutant cells carrying a plasmid encoding TEF2-GSM1-myc (pZL3459) or carrying the empty plasmid (Vector, pRS415TEF) were serially diluted and spotted on YPD (dextrose) and YPL (lactate) medium. Pictures of the plates were taken after 2–4 days’ growth at 30 °C.
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Table 1. Yeast strains used in this study.
Table 1. Yeast strains used in this study.
StrainGenotypeSource
BY4741 (WT)MATa ura3 leu2 his3 met15Lab stock
ZLY2811 (hap4)BY4741 hap4::kanMX4[34]
MBY123 (gsm1)BY4741 gsm1::kanMX4This study
ZLY3707 (adr1)BY4741 adr1::kanMX4[34]
ZLY3701 (cat∆)BY4741 cat8::kanMX4[34]
ZLY5048 (adr1 cat8)BY4741 adr1::kanMX4 cat8::HIS3This study
ZLY5103 (adr1 gsm1)MATa ura3 leu2 his3 lys2 adr1::kanMX4 gsm1::kanMX4This study
ZLY5081 (cat8 gsm1)BY4741 gsm1::kanMX4 cat8::HIS3This study
ZLY5109 (adr1 cat8 gsm1)MATa ura3 leu2 his3 lys2 adr1::kanMX4 gsm1::kanMX4 cat8::HIS3This study
Table 2. Plasmids used in this study.
Table 2. Plasmids used in this study.
PlasmidDescriptionSource
pZL3454pGSM1-lacZ expressing lacZ under the control of a 924 bp GSM1 promoter in the centromeric plasmid WEJ derived from pRS416 [35].This study
pZL3462pRS416-GSM1-GFP expressing Gsm1 from its own promoter with a GFP tag at the C-terminus.This study
pMB165pGSM1(S1M)-lacZ expressing lacZ under the control of a GSM1 promoter with a mutation in the CCAAT sequence at position –634 in relation to the ATG start codon.This study
pMB168pGSM1(S2M)-lacZ expressing lacZ under the control of a GSM1 promoter with a mutation in the CCAAT sequence at position −286 in relation to the ATG start codon.This study
pMB179pFBP1-lacZ expressing lacZ under the control of a 924 bp FBP1 promoter.This study
pZL3628pPCK1-lacZ expressing lacZ under the control of a 1484 bp PCK1 promoter.This study
pMB209pSFC1-lacZ expressing lacZ under the control of a 1454 bp SFC1 promoter.This study
pMB181pLPX1-lacZ expressing lacZ under the control of a 1039 bp LPX1 promoter.This study
pZL3625pYAT1-lacZ expressing lacZ under the control of a 1555 bp YAT1 promoter.This study
pDC124pHAP4-lacZ expressing lacZ under the control of a 1.8 kbp HAP4 promoter.[36]
pZL3613pRS415-GSM1-GFP expressing Gsm1 from its own promoter with a GFP tag at the C-terminus.This study
pZL3459pRS415-TEF2-GSM1-myc, expressing Gsm1 with a C-terminal 3x myc epitope tag under the control of the strong TEF2 promoter. The plasmid was constructed from pRS415TEF [37]. This study
pRS415TEFpRS415-TEF2p-CYC1 terminator.[37]
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Bhondeley, M.; Liu, Z. GSM1 Requires Hap4 for Expression and Plays a Role in Gluconeogenesis and Utilization of Nonfermentable Carbon Sources. Genes 2024, 15, 1128. https://doi.org/10.3390/genes15091128

AMA Style

Bhondeley M, Liu Z. GSM1 Requires Hap4 for Expression and Plays a Role in Gluconeogenesis and Utilization of Nonfermentable Carbon Sources. Genes. 2024; 15(9):1128. https://doi.org/10.3390/genes15091128

Chicago/Turabian Style

Bhondeley, Manika, and Zhengchang Liu. 2024. "GSM1 Requires Hap4 for Expression and Plays a Role in Gluconeogenesis and Utilization of Nonfermentable Carbon Sources" Genes 15, no. 9: 1128. https://doi.org/10.3390/genes15091128

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