Abstract
BackgroundPost-translational modification regulates promoter-binding by Adr1, a Zn-finger transcriptional activator of glucose-regulated genes. Support for this model includes the activation of an Adr1-dependent gene in the absence of Adr1 protein synthesis, and a requirement for the kinase Snf1 for Adr1 DNA-binding. A fusion protein with the Adr1 DNA-binding domain and a heterologous activation domain is glucose-regulated, suggesting that the DNA binding region is the target of regulation.Methodology/Principal FindingsPeptide mapping identified serine 98 adjacent to the Zn-fingers as a phosphorylation site. An antibody specific for phosphorylated serine 98 on Adr1 showed that the level of phosphorylated Adr1 relative to the level of total Adr1 decreased with glucose derepression, in a Snf1-dependent manner. Relative phosphorylation decreased in a PHO85 mutant, and this mutant constitutively expressed an Adr1-dependent reporter. Pho85 did not phosphorylate Adr1 in vitro, suggesting that it affects Adr1 indirectly. Mutation of serine 98 to the phosphomimetic amino acid aspartate reduced in vitro DNA-binding of the recombinant Adr1 DNA-binding domain. Mutation to aspartate or alanine affected activation of a reporter by full-length Adr1, and in vivo promoter binding.Conclusions/SignificanceMutation of Adr1 serine 98 affects in vitro and in vivo DNA binding, and phosphorylation of serine 98 in vivo correlates with glucose availability, suggesting that Adr1 promoter-binding is regulated in part by serine 98 phosphorylation.
Highlights
Glucose repression ensures that yeast cells use preferred carbon sources until available supplies are exhausted
Because phosphorylation is a common mechanism of posttranslational modification, and the Adr1 DBD appears to be crucial to glucose repression, we looked for phosphorylation of the Adr1 DBD as a possible mode of regulation
N-terminal truncations of the Adr1 DBD showed that the site of phosphorylation was within amino acids 94–160, which includes the Cterminal portion of the proximal accessory region (PAR) and the Zn-fingers (Figure 1C, D)
Summary
Glucose repression ensures that yeast cells use preferred carbon sources until available supplies are exhausted. Control of Adr occurs at several levels: through transcription of its gene [5]; post-translational modification of the protein [6]; and access to, or ability to stably bind promoters [7,8] The latter two mechanisms appear to be the most critical, because in glucose-repressed cells, raising Adr protein levels to be comparable to those in derepressed cells does not activate transcription of ADH2 or other Adr1-regulated genes [6,9]. Snf is the yeast homolog of the AMP-activated protein kinase, and activation of Snf in repressing conditions relieves some glucose repression of ADH2 and other Adr1-dependent genes, allowing up to 10% of derepressed transcription levels [8,10]. Post-translational modification regulates promoter-binding by Adr, a Zn-finger transcriptional activator of glucose-regulated genes Support for this model includes the activation of an Adr1-dependent gene in the absence of Adr protein synthesis, and a requirement for the kinase Snf for Adr DNA-binding. A fusion protein with the Adr DNA-binding domain and a heterologous activation domain is glucose-regulated, suggesting that the DNA binding region is the target of regulation
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