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The Saccharomyces cerevisiae MADS-box transcription factor Rlm1 is a target for the Mpk1 mitogen-activated protein
1Transcription Laboratory, ICRF Laboratories, London, United Kingdom.
Abstract:
Mutation of Saccharomyces cerevisiae RLM1, which encodes a MADS-box transcription factor, confers resistance to the toxic effects of constitutive activity of the Mpk1 mitogen-activated kinase (MAPK) pathway. The Rlm1 DNA-binding domain, which is similar to that of the metazoan MEF2 transcription factors, is also closely related to that of a second S. cerevisiae protein, Smp1 (second MEF2-like protein), encoded by the YBR182C open reading frame (N. Demolis et al., Yeast 10:1511-1525, 1994; H. Feldmann et al., EMBO J. 13:5795-5809, 1994). We show that Rlm1 and Smp1 have MEF2-related DNA-binding specificities: Rlm1 binds with the same specificity as MEF2, CTA(T/A)4TAG, while SMP1 binds a more extended consensus sequence, ACTACTA(T/A)4TAG. The two DNA-binding domains can heterodimerize with each other and with MEF2A. Deletion of RLM1 enhances resistance to cell wall disruptants, increases saturation density, reduces flocculation, and inactivates reporter genes controlled by the Rlm1 consensus binding site. Deletion of SMP1 neither causes these phenotypes nor enhances the Rlm1 deletion phenotype. However, overexpression of the DNA-binding domain of either protein causes an osmoremedial phenotype. Synthetic and naturally occurring MEF2 consensus sequences exhibit strong RLM1- and MPK1-dependent upstream activation sequence activity. Transcriptional activation by Rlm1 requires its C-terminal sequences, and Gal4 fusion proteins containing Rlm1 C-terminal sequences also act as MPK1-dependent transcriptional activators. These results establish the Rlm1 C-terminal sequences as a target for the Mpk1 MAPK pathway.
Insights
Mutation in Saccharomyces cerevisiae RLM1 provides resistance to the Mpk1 mitogen-activated protein kinase (MAPK) pathway. RLM1 and Smp1 proteins bind DNA similarly to MEF2 transcription factors, with Rlm1 C-terminal sequences acting as a target for Mpk1.
Area of Science:
- Molecular Biology
- Yeast Genetics
Background:
- Saccharomyces cerevisiae RLM1 encodes a MADS-box transcription factor.
- RLM1's DNA-binding domain is similar to metazoan MEF2 transcription factors and S. cerevisiae Smp1.
- The Mpk1 mitogen-activated protein kinase (MAPK) pathway regulates cell wall integrity.
Purpose of the Study:
- To investigate the DNA-binding specificities of Rlm1 and Smp1.
- To determine the role of Rlm1 and Smp1 in response to Mpk1 pathway activity.
- To identify the Mpk1 pathway's target sequences within Rlm1.
Main Methods:
- DNA-binding specificity assays for Rlm1 and Smp1.
- Analysis of phenotypes associated with RLM1 and SMP1 deletion mutants.
- Reporter gene assays to assess transcriptional activation.
- Gal4 fusion protein experiments to map functional domains.
Main Results:
- Rlm1 and Smp1 exhibit MEF2-related DNA-binding specificities, with distinct consensus sequences.
- Deletion of RLM1 confers resistance to cell wall disruptants and alters cell density and flocculation.
- Overexpression of DNA-binding domains induces an osmoremedial phenotype.
- Rlm1 C-terminal sequences are required for transcriptional activation and are targeted by the Mpk1 MAPK pathway.
Conclusions:
- Rlm1 and Smp1 are transcription factors with MEF2-like DNA-binding properties.
- The RLM1 gene plays a significant role in cellular responses to cell wall stress.
- The Mpk1 MAPK pathway directly regulates Rlm1 activity via its C-terminal domain.