GSM1 Requires Hap4 for Expression and Plays a Role in Gluconeogenesis and Utilization of Nonfermentable Carbon Sources
Abstract
:1. Introduction
2. Materials and Methods
2.1. Growth Media, Growth Conditions, Strains, and Plasmids
2.2. Yeast Transformation and β-Galactosidase Activity Assays
2.3. Serial Dilution of Cells for Growth Analysis
2.4. Cellular Extract Preparation, Immunoblotting, and Immunoprecipitation
3. Results
3.1. GSM1 Expression Is Subject to Glucose Repression and Requires Hap4 under Glucose-Derepression Conditions
3.2. A Proximal CCAAT Sequence Element Is Required for GSM1 Expression
3.3. Identification of Potential Gsm1 Target Genes via Analysis of lacZ Reporter Genes
3.4. GSM1 Overexpression Increases Expression of FBP1-, LPX1-, PCK1-, SFC1-, and YAT1-lacZ Reporter Genes in hap4∆ Mutant Cells
3.5. Cat8 and Gsm1 Are Important in the Transcriptional Regulation of FBP1, PCK1, SFC1, and YAT1 and in the Utilization of Lactate
3.6. GSM1 Overexpression Increases Target-Gene Expression in adr1∆ cat8∆ Mutant Cells and Suppresses Growth Defects Associated with cat8Δ
4. Discussion
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Strain | Genotype | Source |
---|---|---|
BY4741 (WT) | MATa ura3 leu2 his3 met15 | Lab stock |
ZLY2811 (hap4) | BY4741 hap4::kanMX4 | [34] |
MBY123 (gsm1) | BY4741 gsm1::kanMX4 | This study |
ZLY3707 (adr1) | BY4741 adr1::kanMX4 | [34] |
ZLY3701 (cat∆) | BY4741 cat8::kanMX4 | [34] |
ZLY5048 (adr1 cat8) | BY4741 adr1::kanMX4 cat8::HIS3 | This study |
ZLY5103 (adr1 gsm1) | MATa ura3 leu2 his3 lys2 adr1::kanMX4 gsm1::kanMX4 | This study |
ZLY5081 (cat8 gsm1) | BY4741 gsm1::kanMX4 cat8::HIS3 | This study |
ZLY5109 (adr1 cat8 gsm1) | MATa ura3 leu2 his3 lys2 adr1::kanMX4 gsm1::kanMX4 cat8::HIS3 | This study |
Plasmid | Description | Source |
---|---|---|
pZL3454 | pGSM1-lacZ expressing lacZ under the control of a 924 bp GSM1 promoter in the centromeric plasmid WEJ derived from pRS416 [35]. | This study |
pZL3462 | pRS416-GSM1-GFP expressing Gsm1 from its own promoter with a GFP tag at the C-terminus. | This study |
pMB165 | pGSM1(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 |
pMB168 | pGSM1(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 |
pMB179 | pFBP1-lacZ expressing lacZ under the control of a 924 bp FBP1 promoter. | This study |
pZL3628 | pPCK1-lacZ expressing lacZ under the control of a 1484 bp PCK1 promoter. | This study |
pMB209 | pSFC1-lacZ expressing lacZ under the control of a 1454 bp SFC1 promoter. | This study |
pMB181 | pLPX1-lacZ expressing lacZ under the control of a 1039 bp LPX1 promoter. | This study |
pZL3625 | pYAT1-lacZ expressing lacZ under the control of a 1555 bp YAT1 promoter. | This study |
pDC124 | pHAP4-lacZ expressing lacZ under the control of a 1.8 kbp HAP4 promoter. | [36] |
pZL3613 | pRS415-GSM1-GFP expressing Gsm1 from its own promoter with a GFP tag at the C-terminus. | This study |
pZL3459 | pRS415-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 |
pRS415TEF | pRS415-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
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 StyleBhondeley, 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