S. cerevisiae uses a complex network of signaling systems and transcriptional regulons to recognize and respond to environmental pressures. We report the transcriptional response of S. cerevisiae to .NO-mediated nitrosative stress. Although treatment with DPTA NONOate led to a general stress response common to other perturbation experiments, DPTA NONOate and .NO gas also induced a genetically separable, physiologically relevant set of genes. This set of genes is comprised of YHB1, SSU1, YNR064c, and YNL335w/YFL061w.
Yhb1p catalyzes the reaction of .
NO with oxygen to create nitrate, limiting the exposure of the cell to .
NO and thus confers a growth advantage after .
NO treatment in YPD media (Liu et al., 2000
). In contrast to previous studies in which S. cerevisiae
was exposed to DETA NONOate (Liu et al., 2000
), nitrosoglutathione (Ullmann et al., 2004
), or sodium nitrite (Ullmann et al., 2004
), we found that both mRNA and protein levels of YHB1
were responsive to .
NO exposure. This discrepancy might be due to insufficient .
NO availability, cytotoxic .
NO effects, media effects, or differences in strain background. In addition, we found that the deletion of the YHB1
gene seems to lead to an increase in the general stress response after DPTA NONOate treatment yet does not affect the induction of SSU1
and the other ORFs in the RNI-responsive gene cluster.
Ssu1p has been reported to be a sulfite efflux transporter and to be located at the outer membrane (Park and Bakalinsky, 2000
). We found that the SSU1
transcript and protein levels increased after nitrosative stress. Furthermore, the presence of the SSU1
gene conferred a growth advantage after exposure to DPTA NONOate in SCD media. We speculate that in addition to transporting sulfite, SSU1
also may transport .
NO metabolites out of the cell.
The predicted ORFs YNR064c and YNL335w/YFL061c also were induced after nitrosative stress. The presence of these genes did not confer significant growth advantages (our unpublished data). The function of these genes with regard to nitrosative stress remains unclear. It is possible that these ORFs may have roles in .NO detoxification that were not revealed by the laboratory conditions used for the growth advantage assay.
FZF1 encodes a Zn-finger DNA binding transcription factor necessary for SSU1 transcription, and until this study, SSU1 was the only known target. We found that the FZF1 gene was required for the induction of YHB1, SSU1, and the other ORFs after nitrosative stress. Furthermore, overexpression led to induction of YHB1, SSU1, and the other uncharacterized ORFs of the RNI-responsive gene cluster. Importantly, the Fzf1p-overexpressing strain exhibited a growth advantage relative to wild type after DPTA NONOate treatment. Deletion of FZF1 resulted in a growth disadvantage that was less profound than the growth disadvantage caused by deletion of YHB1 in YPD media, or deletion of SSU1 in SCD media. This is likely due to FZF1-independent basal transcription of YHB1 and SSU1.
Overexpression of Fzf1p resulted in increased transcription of the target genes, despite FZF1 mRNA levels remaining constant after DPTA treatment. The mechanistic explanation for these observations may be similar to the regulation of Pho4p and other transcription factors for which phosphorylation or some other posttranslational modification leads to a differential subcellular localization. The situation could be analogous in that the overexpression of Fzf1p may stoichiometrically outcompete a regulatory mechanism that would ultimately result in transcriptional activation.
Although it may be that additional components are required for sensing .
NO or .
NO-derived metabolites before activation of Fzf1p, it also may be the case that these capabilities are inherent to Fzf1p alone. It is plausible that nitrosylation of Fzf1p could lead to its modulation as an activator. Interestingly, induction of the FZF1
-dependent gene set did not occur after treatment with the thiol oxidant diamide, heat shock, or in other oxidative stresses (Gasch et al., 2000
Previous studies have shown that after treatment with methyl methane sulfonate (MMS), SSU1
, YNR064c, and YNL335w/YFL061c are induced raising the possibility that DNA damage also may activate the FZF1
-dependent gene set. However, YHB1
mRNA was not significantly induced after this treatment (Gasch et al., 2001
), and a genome-wide screen of the deletion library for MMS hypersensitivity did not find that ssu1
Δ or fzf1
Δ strains were sensitive (Chang et al., 2002
It has previously been reported that Fzf1p specifically binds the SSU1
promoter in vitro (Avram et al., 1999
). The conserved sequence motif CS1 is contained within the region protected from DNAse I cleavage, yet this sequence could not be located in the promoter regions of the other .
NO-responsive genes. Sequence comparisons revealed a CS2 in the promoters of the FZF1
-dependent gene set that was sufficient for DPTA NONOate-mediated, FZF1
-dependent induction. These data imply that Fzf1p possesses the ability to interact with at least two distinct consensus binding sequences, given that CS1 and CS2 have no obvious similarity. Because we have not shown a direct biochemical interaction between CS2 and Fzf1p, it is formally possible that induction via CS2 is an indirect effect of Fzf1p action. Further in vitro and in vivo DNA binding studies will directly address this issue.
The growth inhibition effect of .NO-mediated stress seems to be partially dependent on environmental factors and understanding the effect of growth conditions is essential for proper interpretation of these assays. In minimal media, 10- to 15-fold less DPTA NONOate or .NO gas was necessary to induce a specific response compared with experiments conducted in YPD. An obvious difference between the composition of YPD and SCD includes higher thiol concentrations, which may account for the differential sensitivity of yeast in these two media.
is a relevant model organism for studying the response to .
NO because pathogenic fungi C. albicans
and C. neoformans
are likely to use similar molecular signaling mechanisms to induce Yhb1p levels in response to .
NO. The orthologous flavohemoglobin in C. albicans
has already been shown to respond to .
NO (Ullmann et al., 2004
), and the C. albicans
genome-wide transcriptional response bares significant similarity to the response we observe in S. cerevisiae
, although the corresponding transcription factor has not been identified (Bethann Hromatka, personal communication). Further dissection of the mechanism by which S. cerevisiae
senses and responds to .
NO may shed light on this important molecule.