The MSN2 and MSN4 genes encode homologous and functionally redundant Cys2His2 zinc finger proteins. A disruption of both MSN2 and MSN4 genes results in a higher sensitivity to different stresses, including carbon source starvation, heat shock and severe osmotic and oxidative stresses. We show that MSN2 and MSN4 are required for activation of several yeast genes such as CTT1, DDR2 and HSP12, whose induction is mediated through stress‐response elements (STREs). Msn2p and Msn4p are important factors for the stress‐induced activation of STRE dependent promoters and bind specifically to STRE‐containing oligonucleotides. Our results suggest that MSN2 and MSN4 encode a DNA‐binding component of the stress responsive system and it is likely that they act as positive transcription factors.

In all living organisms, appropriate reactions to unfavorable environmental conditions (stress factors) are observed. When transcription in eukaryotic cells is controlled by extracellular signals, at least one signaling component has to be translocated from the cytoplasm to the nucleus in a signal-dependent manner. The signaling components may be of low molecular weight (second messengers) or protein members of the signaling cascades. Examples for the latter are MAP kinases, for example, the p42 MAP kinase and p44 ERK1 on mitogenic stimulation (Chen et al. 1992).Regulated nuclear translocation is also found widely at the level of transcription factors and is achieved by either cytoplasmic anchoring or activation of nuclear localization signals (NLS) by unmasking or modification (Jans 1995;Gö rlich and Mattaj 1996;Nigg 1997). A prominent example of this type of control is provided by NF-B. A rapid transcriptional response to a variety of stress stimuli is elicited by phosphorylation of the inhibitory factor IB followed by its dissociation from the transcription factor and destruction. This leads to unmasking of the NF-B nuclear localization signal and to the subsequent translocation to the nucleus (Siebenlist, et al. 1995;Baldwin 1996). Another example is NF-ATc, a transcription factor involved in early immune responses that is translocated to the nucleus on dephosphorylation by Ca 2+

The transcriptional response to environmental changes is a major topic in both basic and applied research. From a basic point of view, to understand this response includes unravelling how the stress signal is sensed and transduced to the nucleus, to identify which genes are induced under each stress condition and, finally, to establish the phenotypic consequences of this induction in stress tolerance. The possibility of using genetic approaches has made the yeast Saccharomyces cerevisiae a compelling model to study stress response at a molecular level. Moreover, this information can be used to isolate and characterise stress-related proteins in higher eukaryotes and to design strategies to increase stress resistance in organisms of industrial interest. In this review the progress made in recent years is discussed.

The transcriptional response to environmental changes is a major topic in both basic and applied research. From a basic point of view, to understand this response includes unravelling how the stress signal is sensed and transduced to the nucleus, to identify which genes are induced under each stress condition and, finally, to establish the phenotypic consequences of this induction in stress tolerance. The possibility of using genetic approaches has made the yeast Saccharomyces cerevisiae a compelling model to study stress response at a molecular level. Moreover, this information can be used to isolate and characterise stress-related proteins in higher eukaryotes and to design strategies to increase stress resistance in organisms of industrial interest. In this review the progress made in recent years is discussed.

In the budding yeast Saccharomyces cerevisiae, the SNF1 (CAT1/CCR1) protein-serine/threonine kinase is required for expression of many genes in response to glucose limitation (6,48). This important signalling pathway is as yet incompletely understood, although a number of proteins that function in the SNF1 pathway have been identified. At least two proteins are physically associated with SNF1 in a multiprotein complex: SNF4 (CAT3), which serves as a positive effector (7,8,16,49), and SIP1, which is phosphorylated in vitro (59). The SNF1 pathway appears to inhibit transcriptional repression mediated by the SSN6, TUP1, and MIG1 proteins (26,33,34,50,58), as mutations in SSN6, TUPI, and MIGJ suppress the requirement for SNF1 (35,56). Further understanding of the regulatory mechanism requires the identification of other components of the SNF1 pathway.To identify proteins that are functionally related to SNF1, we have sought genes that in increased dosage compensate for defects in SNF1 protein kinase function caused by temperature-sensitive snfl (snfl-ts) (4,10,18,24,27,30,54). MATERUILS AND METHODSStrains and genetic methods. Strains of S. cerevisiae used in this study are listed in Table 1. Standard methods were used for genetic analysis and transformation (42). Growth on different carbon sources was scored by spotting cell suspensions or by streaking cells on solid medium. Anaerobic growth on raffinose and galactose was scored by incubating plates in GasPaks (BBL).Isolation of temperature-sensitive alleles of SNFI. The centromere-containing plasmid pCE101 carrying the SNFI gene on a XhoI-BamHI fragment (7) was mutagenized with hydroxylamine (40) and used to transform MCY1845 (snfl-AIO). Ura+ transformants (-20,000) were replicated to supplemented synthetic (SD) medium lacking uracil and containing 2% raffinose (SR-Ura). The plates were incubated at 37°C, and 219 transformants that were unable to grow were retested for growth on SR-Ura at 25, 30, and 37°C. Ten showed a temperature-sensitive (Ts) phenotype, and plasmid DNA recovered from five of them conferred the Ts phenotype. The fragment containing the SNF gene was recloned in YCp5O (41)

In response to various stresses, as well as during the diauxic transition, the Msn2p and Msn4p transcription factors of Saccharomyces cerevisiae are activated and induce a large set of genes. This activation is inhibited by the Ras/cAMP/PKA (cAMP-dependent protein kinase) pathway. Here we show by immunoblotting experiments that Msn2p and Msn4p are phosphorylated in vivo during growth on glucose, and become hyperphosphorylated at the diauxic transition and upon heat shock. This hyperphosphorylation is correlated with activation of Msn2/4p-dependent transcription. An increased level of cAMP prevents and reverses these hyperphosphorylations, indicating that kinases other than PKA are involved. These results suggest that PKA and stress-activated kinases control Msn2/4p activity by antagonistic phosphorylation. It was also noted that Msn4p is transiently increased at the diauxic transition. Msn2p and Msn4p present different hyperphosphorylation patterns in response to different stresses.

SummaryIn this report we show that the ENA1/PMR2A gene is under glucose repression. The SNF1 protein kinase, acting independently from the HOG and calcineurin pathways, is essential to release ENA1 from glucose repression. The transcriptional repressor Ssn6p negatively regulates ENA1 expression and, like other glucose repressible genes, this repression is mediated in part by Mig1p. Deletion of a fragment from the ENA1 promoter that includes two Mig1p consensus binding sites gives a high level of expression in glucose without added salt. We suggest that regulation of ENA1 by the SNF1 pathway could be part of a general mechanism through which yeast cells respond to carbon source starvation by activating protective systems against different types of stress.

SummarySaccharomyces cerevisiae possesses several transcription factors involved in the transcriptional activation of stress-induced genes. Among them, the heat shock factor (Hsf1p) and the zinc finger proteins of the general stress response (Msn2p and Msn4p) have been shown to play a major role in stress protection. Some heat shock protein (HSP) genes contain both heat shock elements (HSEs) and stress response elements (STREs), suggesting the involvement of both transcription factors in their regulation. Analysis of the stress-induced expression of two of these genes, HSP26 and HSP104, reveals that the contribution of Hsf1p and Msn2/4p is different depending on the gene and the stress condition.