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

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.

Keywords: Cat8; Fbp1; Gsm1; Hap2/3/4/5; Hap4; Pck1; S. cerevisiae; gluconeogenesis; transcriptional regulation.

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 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 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 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 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 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.