RESUMO
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.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Parede Celular/metabolismo , Sequência Consenso , DNA Fúngico/genética , DNA Fúngico/metabolismo , Dimerização , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Deleção de Genes , Genes Fúngicos , Proteínas de Domínio MADS , Dados de Sequência Molecular , Mutação , Fenótipo , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Transativadores/química , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
The MEF2 proteins are involved in regulation of many muscle specific genes. Although MEF2 RNAs encoding the MEF2A and MEF2D isoforms are ubiquitously expressed, the presence of MEF2 proteins in non-muscle cell types has been controversial. Here we use a well-characterised antibody in conjunction with DNA binding studies to provide evidence that members of the MEF2 family are widely expressed in the nuclei of cultured cells and are competent to bind DNA. The data show that non-muscle MEF2 complexes contain MEF2A, and that another MEF2 protein, probably MEF2D, is also present. These results suggest that MEF2 proteins fulfil functions in addition to muscle-specific gene expression.
