The Saccharomyces cerevisiae GATA-factor activators' response to the 14-3-3 proteins, nutrient limitation, rapamycin and MSX treatment, and cis-acting factors

The ability of Saccharomyces cerevisiae to mount quick and precise responses to the ever changing environmental conditions it faces is critical to its survival. S. cerevisiae cells utilize the GATA-factor transcriptional activators Gln3 and Gat1 to mediate expression of NCR-sensitive genes when faced with nitrogen stress. The goal of this work is to further examine these GATA-factor activators and to expand the body of knowledge that encompasses their states of phosphorylation, intracellular localization, and DNA binding requirements.; The 14-3-3 proteins are a conserved family of acidic dimeric proteins present in all eukaryotes, and implicated as having in excess of 150 binding partners. The S. cerevisiae 14-3-3 proteins, Bmh1 and Bmh2, are shown here to affect the NCR system, with the following data in a bmh1Deltabmh2Delta mutant: (i) decreased yet normally-regulated levels of NCR-sensitive transcription (ii) altered states of Gln3-Myc13 phosphorylation, and (iii) decreased ability of Gln3-Myc13 to localize in the nucleus.; Gln3 has been shown previously to respond, via phosphorylation and intracellular localization, to multiple changes in its environment: (i) rapamycin, the TOR inhibiting antineoplastic drug (ii) MSX, a glutamine synthetase inhibitor, and (iii) to carbon and nitrogen starvation. We compared the response of Gat1 and Gln3 to these conditions. Intracellular localization of Gat1 and Gln3 correlates with the transcription they mediate. In contrast, three characteristics of Gat1 and Gln3 differ significantly: (i) the kinetics of their localization in response to nutritional transitions (nitrogen and carbon starvation) and rapamycin-treatment, (ii) their opposite localization responses to MSX-treatment, and (iii) their phosphorylation levels in the above situations.; Gln3 and Gat1 share a highly homologous zinc-finger DNA-binding domain. It is known that they both bind to sequences containing 5'GATAA 3' at their core. Two well studied NCR-sensitive genes, DAL5 and GAP1, were investigated via promoter analysis. The data indicate: (i) one (UASB) and possibly two (UAS A) additional cis-acting elements are required for full DAL5 expression, (ii) only two of the GAP1 promotor GATA elements are capable of supporting both Gln3 and Gat1 mediated expression (iii) Gln3 and Gat1 mediated expression of a saturation mutagenized GATA-promoter element illustrates their differing sequence preferences for transcriptional activation.