Redox control of AP-1-like factors in yeast and beyond.

Cells have evolved complex and efficient strategies for dealing with variable and often-harsh environments. A key aspect of these stress responses is the transcriptional activation of genes encoding defense and repair proteins. In yeast members of the AP-1 family of proteins are required for the transcriptional response to oxidative stress. This sub-family of AP-1 (called yAP-1) proteins are sensors of the redox-state of the cell and are activated directly by oxidative stress conditions. yAP-1 proteins are bZIP-containing factors that share homology to the mammalian AP-1 factor complex and bind to very similar DNA sequence sites. The generation of reactive oxygen species and the resulting potential for oxidative stress is common to all aerobically growing organisms. Furthermore, many of the features of this response appear to be evolutionarily conserved and consequently the study of model organisms, such as yeast, will have widespread utility. The important structural features of these factors, signaling pathways controlling their activity and the nature of the target genes they control will be discussed.

AP-1 transcription factors in yeast.

In the past two years, the completion of the Saccharomyces cerevisiae genome project and molecular analysis of other fungal species has resulted in the identification of a growing number of yeast AP-1 transcription factors. Characterisation of these factors indicates that, like their mammalian counterparts, they activate gene expression in response to a variety of extracellular stimuli. In particular, these factors are required for the response to oxidative stress and for surviving exposure to a variety of cytotoxic agents. Much progress has also been made in understanding how members of this family of proteins are regulated. These studies promise to further our awareness of eukaryotic stress responses and are likely to have implications for the study of mammalian AP-1.

Stress-activated signalling pathways in yeast.

Eukaryotic cells have developed response mechanisms to combat the harmful effects of a variety of stress conditions. In the majority of cases, such responses involve changes in the gene expression pattern of the cell, leading to increased levels and activities of proteins that have stress-protective functions. Over the last few years, considerable progress has been made in understanding how stress-dependent transcriptional changes are brought about, and it transpires that the underlying mechanisms are highly conserved, being similar in organisms ranging from yeast to man. Many of the stress signals derive from the extracellular environment and accordingly these signals require transduction from the cell surface to the nucleus. This is accomplished through stress-activated signalling pathways, key amongst which are the highly conserved stress-activated MAP kinase pathways. Stimulation of these pathways leads to the increased activity of specific transcription factors and consequently the increased expression of certain stress-related genes. In this review, we focus on the progress that has been made in understanding these stress responses in yeast.

Regulation of the fission yeast transcription factor Pap1 by oxidative stress: requirement for the nuclear export factor Crm1 (Exportin) and the stress-activated MAP kinase Sty1/Spc1.

The fission yeast Sty1 stress-activated MAP kinase is crucial for the cellular response to a variety of stress conditions. Accordingly, sty1- cells are defective in their response to nutrient limitation, lose viability in stationary phase, and are hypersensitive to osmotic stress, oxidative stress, and UV treatment. Some of these phenotypes are caused by Sty1-dependent regulation of the Atf1 transcription factor, which controls both meiosis-specific and osmotic stress-responsive genes. However, in this report we demonstrate that the cellular response to oxidative stress and to treatment with a variety of cytotoxic agents is the result of Sty1 regulation of the Pap1 transcription factor, a bZip protein with structural and DNA binding similarities to the mammalian c-Jun protein. We show that both Sty1 and Pap1 are required for the expression of a number of genes involved in the oxidative stress response and for the expression of two genes, hba2+/bfr1+ and pmd1+, which encode energy-dependent transport proteins involved in multidrug resistance. Furthermore, we demonstrate that Pap1 is regulated by stress-dependent changes in subcellular localization. On imposition of oxidative stress, the Pap1 protein relocalizes from the cytoplasm to the nucleus in a process that is dependent on the Sty1 kinase. This relocalization is the result of regulated protein export, rather than import, and involves the Crm1 (exportin) nuclear export factor and the dcd1+/pim1+ gene that encodes an Ran nucleotide exchange factor.

A genetic screen reveals a role for the late G1-specific transcription factor Swi4p in diverse cellular functions including cytokinesis.

The transcription factor Swi4p plays a crucial role in the control of the initiation of the cell cycle in budding yeast. To further understand Swi4p function, we set up a synthetic lethal screen for genes interacting with SWI4. Fourteen conditional mutations which resulted in lethality only in the absence of SWI4 have been isolated. Only two of them were suppressed by ectopic expression of CLN2, indicating that Swi4p is involved in diverse cellular processes in addition to its requirement for CLN1,2 regulation. In most of the mutants a cell cycle phenotype was observed, including defects in G1 progression, budding, the G2/M transition and cytokinesis. In addition, four of the mutations resulted in massive cell lysis at the restrictive temperature, indicating that Swi4p is involved in the maintenance of cell integrity. One of the mutants, rsf1 swi4delta, was characterized in detail and it is defective in cytokinesis at the restrictive temperature. Staining with Calcofluor revealed that the rsf1 swi4delta mutant is impaired in chitin biosynthesis. rsf1 is allelic to the AGM1 gene, coding for N-acetylglucosamine-phosphate mutase, an enzyme involved in the biosynthesis of chitin. A single copy of SWI4 suppressed the cytokinesis defect. The above data suggest that Swi4p has a role in cytokinesis and becomes essential in this process when chitin biosynthesis is compromised. As overexpression or ectopic expression of CLN did not suppress the rsf1 swi4delta mutant phenotype, Swi4p must control some other gene(s) involved in cytokinesis.

The Skn7 response regulator controls gene expression in the oxidative stress response of the budding yeast Saccharomyces cerevisiae.

Deletion of the bacterial two-component response regulator homologue Skn7 results in sensitivity of yeast to oxidizing agents indicating that Skn7 is involved in the response to this type of stress. Here we demonstrate that following oxidative stress, Skn7 regulates the induction of two genes: TRX2, encoding thioredoxin, and a gene encoding thioredoxin reductase. TRX2 is already known to be induced by oxidative stress dependent on the Yap1 protein, an AP1-like transcription factor responsible for the induction of gene expression in response to various stresses. The thioredoxin reductase gene has not previously been shown to be activated by oxidative stress and, significantly, we find that it too is regulated by Yap1. The control of at least TRX2 by Skn7 is a direct mechanism as Skn7 binds to the TRX2 gene promoter in vitro. This shows Skn7 to be a transcription factor, at present the only such eukaryotic two-component signalling protein. Our data further suggest that Skn7 and Yap1 co-operate on the TRX2 promoter, to induce transcription in response to oxidative stress.

The Swi5 transcription factor of Saccharomyces cerevisiae has a role in exit from mitosis through induction of the cdk-inhibitor Sic1 in telophase.

Deactivation of the B cyclin kinase (Cdc28/Clb) drives the telophase to G1 cell cycle transition. Here we investigate one of the control pathways than contributes to kinase deactivation, involving the cell cycle-regulated production of the cdk inhibition Sic1. We show that the cell cycle timing of SIC1 expression depends on the transcription factor Swi5, and that Swi5-dependent SIC1 expression begins during telophase. In contrast to Swi5, the related transcription factor Ace2, which can also induce SIC1 expression, is not active during telophase. The functional consequence of Swi5-regulated SIC1 expression in vivo is that both sic1 delta and swi5 delta strains have identical mitotic exit-related phenotypes. First, both are synthetically lethal with dbj2 delta, resulting in cell cycle arrest in telophase. Second, both are hypersensitive to overexpression of the B cyclin CLB2. Thus Swi5-dependent activation of the SIC1 gene contributes to the deactivation of the B cyclin kinase, and hence exit from mitosis.

Getting started: regulating the initiation of DNA replication in yeast.

Initiation of DNA replication in yeast appears to operate through a two-step process. The first step occurs at the end of mitosis in the previous cell cycle, where, following the decrease in B cyclin-dependent kinase activity, an extended protein complex called the prereplicative complex (pre-RC) forms over the origin of replication. This complex is dependent on the association of the Cdc6 protein with the Origin Recognition Complex (ORC) and appears concomitantly with the nuclear entry of members of the Mcm family of proteins. The second step is dependent upon the cell passing through a G1 decision point called Start. If the environmental conditions are favorable, and the cells reach a critical size, then there is a rise in G1 cyclin-dependent kinase activity, which leads to the activation of downstream protein kinases; the protein kinases are, in turn, required for triggering initiation from the preformed initiation complexes. These protein kinases, Dbf4-Cdc7 and Clb5/6(B-cyclin)-Cdc28, are thought to phosphorylate targets within the pre-RC. The subsequent rise in B cyclin protein kinase activity following Start not only triggers origin firing, but also inhibits the formation of new pre-RCs, which ensures that there is only one S phase in each cell cycle. The destruction of B-cyclin protein kinase activity at the end of the cell cycle potentiates the formation of new pre-RCs-resetting origins for the next S phase.

The Atf1 transcription factor is a target for the Sty1 stress-activated MAP kinase pathway in fission yeast.

The atf1+ gene of Schizosaccharomyces pombe encodes a bZIP transcription factor with strong homology to the mammalian factor ATF-2. ATF-2 is regulated through phosphorylation in mammalian cells by the stress-activated mitogen-activated protein (MAP) kinases SAPK/JNK and p38. We show here that the fission yeast Atf1 factor is also regulated by a stress-activated kinase, Sty1. The Sty1 kinase is stimulated by a variety of different stress conditions including osmotic and oxidative stress and heat shock. Deletion of the atf1+ gene results in many, but not all, of the phenotypes associated with loss of Sty1, including sensitivity to environmental stress and inability to undergo sexual conjugation. Furthermore, we identify a number of target genes that are induced rapidly in a manner dependent upon both the Sty1 kinase and the Atf1 transcription factor. These genes include gpd1+, which is important for the response of cells to osmotic stress, the catalase gene lambda important for cells to combat oxidative stress, and pyp2+, which encodes a tyrosine-specific MAP kinase phosphatase. Induction of Pyp2 by Atf1 is direct in that it does not require de novo protein synthesis and results in a negative feedback loop that serves to control signaling through the Sty1/Wis1 pathway. We show that Atf1 associates stably and is phosphorylated by the Sty1 kinase in vitro. Taken together, these results indicate that the interaction between AM and Sty1 is direct. These findings highlight a remarkable level of conservation in transcriptional control by stress-activated MAP kinase pathways between fission yeast and mammalian cells.

Rme1, a negative regulator of meiosis, is also a positive activator of G1 cyclin gene expression.

Control of G1 cyclin expression in Saccharomyces cerevisiae is mediated primarily by the transcription factor SBF (Swi4/Swi6). In the absence of Swi4 and Swi6 cell viability is lost, but can be regained by ectopic expression of the G1 cyclin encoding genes, CLN1 or CLN2. Here we demonstrate that the RME1 (regulator of meiosis) gene can also bypass the normally essential requirement for SBF. RME1 encodes a zinc finger protein which is able to repress transcription of IME1 (inducer of meiosis) and thereby inhibit cells from entering meiosis. We have found that expression of RME1 from a high copy number plasmid can specifically induce CLN2 expression. Deletion of RME1 alone shows no discernible effect on vegetative growth, however, deletion of RME1 in a swi6 delta swi4ts strain results in a lowering of the non-permissive temperature for viability. This suggests that Rme1 plays a significant but ancillary role in SBF in inducing CLN2 expression. We show that Rme1 interacts directly with the CLN2 promoter and have mapped the region of the CLN2 promoter required for Rme1-dependent activation. Consistent with Rme1 having a cell cycle role in G1, we have found that RME1 mRNA is synthesized periodically in the cell cycle, with maximum accumulation occurring at the M/G1 boundary. Thus Rme1 may act both to promote mitosis, by activating CLN2 expression, and prevent meiosis, by repressing IME1 expression.

HIV-1 Tat directly interacts with the interferon-induced, double-stranded RNA-dependent kinase, PKR.

We present evidence that the HIV-1 Tat protein and the RNA-dependent cellular protein kinase, PKR, interact with each other both in vitro and in vivo. Using GST fusion chromatography, we demonstrate that PKR, interacts directly with the HIV-1 Tat protein. The region in Tat sufficient for binding PKR maps within amino acids 20 to 72. In in vitro assays, the two-exon form of Tat (Tat 86) was phosphorylated by PKR, while the one exon form of Tat (Tat 72) inhibited PKR autophosphorylation and substrate phosphorylation. The ability of Tat to interact with PKR was demonstrated in both yeast and mammalian cells. Expression of PKR in yeast results in a growth suppressor phenotype which was reversed by coexpression of a one exon form of Tat. Expression of Tat 72 in HeLa cells resulted in direct interaction with PKR as detected by coimmunprecipitation with a Tat antibody. Tat and PKR also form a coimmunoprecipitable complex in cell-free extracts prepared from productively infected T lymphocytes. The interaction of Tat with PKR provides a potential mechanism by which HIV could suppress the interferon system.

The activation of DNA replication in yeast.

DNA replication in eukaryotic cells is initiated at sites in the DNA known as origins. Studies in yeast have identified a number of the genes and proteins that may be involved in this process. In this review, we concentrate largely on those genes on proteins that are required for initiation of DNA replication and for which there is some evidence for a role at origins.

Mutational analysis of the double-stranded RNA (dsRNA) binding domain of the dsRNA-activated protein kinase, PKR.

The interferon-induced, double-stranded RNA (dsRNA)-dependent protein kinase, PKR, is an inhibitor of translation and has antiviral, antiproliferative, and antitumor properties. Previously, the dsRNA binding domain had been located within the N-terminal region of PKR and subsequently shown to include two nearly identical domains comprising residues 55-75 and 145-166. We have undertaken both random and site-directed, alanine-scanning mutagenesis in order to investigate the contribution of individual amino acids within these domains to dsRNA binding. Here we identify 2 residues that were absolutely required for dsRNA binding, glycine 57 and lysine 60. Mutation of 2 other residues within the domain (lysine 64 and leucine 75) resulted in less than 10% binding (compared to wild type). We have also identified a number of other residues that influence dsRNA binding to varying degrees. Mutants that were unable to bind dsRNA were not active in vitro and possessed no antiproliferative activity in vivo. However, dsRNA binding mutants were partially transdominant over wild type PKR in mammalian cells, suggesting that binding of dsRNA activator is not the mechanism responsible for the phenotype of PKR mutants.

Mutations causing aminotriazole resistance and temperature sensitivity reside in gyrB, which encodes the B subunit of DNA gyrase.

Certain mutations in gyrA and gyrB, the genes encoding the two subunits of DNA gyrase, are known to influence expression of the his operon (K. E. Rudd and R. Menzel, Proc. Natl. Acad. Sci. USA 84:517-521, 1987). Such mutations lead to a decrease in tRNA(His) levels and consequently to an attenuator-dependent increase in his operon expression. This effect presumably is due to the dependence of the hisR promoter (hisR encodes tRNA(His) on supercoiling for maximal activity. We used a relaxed (Rel-) strain of Escherichia coli to isolate gyrB mutants by selecting for resistance to the histidine antimetabolite 3-amino-1,2,4-triazole and then screening for temperature-sensitive growth on rich medium. Rel- mutants, which generally have lower basal levels of ppGpp (a positive regulator of his operon transcription), are more sensitive than wild-type E. coli to aminotriazole. The chance of isolating spoT mutants, which can be selected with a similar procedure, was decreased by selecting in the presence of a multicopy plasmid that carries the wild-type spoT gene. Under these conditions, gyrB mutants were isolated preferentially. This scheme selects for loss of function of DNA gyrase, rather than for its alteration due to resistance to specific gyrase inhibitors, and thus a greater variety of gyrase mutations might be obtainable.

deaD, a new Escherichia coli gene encoding a presumed ATP-dependent RNA helicase, can suppress a mutation in rpsB, the gene encoding ribosomal protein S2.

We have cloned and sequenced a new gene from Escherichia coli which encodes a 64-kDa protein. The inferred amino acid sequence of the protein shows remarkable similarity to eIF4A, a murine translation initiation factor that has an ATP-dependent RNA helicase activity and is a founding member of the D-E-A-D family of proteins (characterized by a conserved Asp-Glu-Ala-Asp motif). Our new gene, called deaD, was cloned as a gene dosage-dependent suppressor of temperature-sensitive mutations in rpsB, the gene encoding ribosomal protein S2. We suggest that the DeaD protein plays a hitherto unknown role in translation in E. coli.

A region of the immunoglobulin-mu heavy chain necessary for forming pentameric IgM.

In order to define the molecular requirements for IgM pentamer formation, we have isolated several mutant hybridomas which produce predominantly monomeric IgM. For one such mutant, 102, we synthesized a cDNA clone of its mu-mRNA, and found an in-frame 39-bp deletion, which thus encodes a mu-chain lacking amino acids 550-562, a region spanning the fourth constant domain and the tail of the mu-chain. To prove that this deletion is sufficient to block pentamer formation, we used site-directed mutagenesis to construct a mu-gene lacking these 39 bp, and have shown that the expression of this altered mu-gene results in the production of monomeric IgM.