PP2ARts1 antagonizes Rck2-mediated hyperosmotic stress signaling in yeast.

In Saccharomyces cerevisiae, impairment of protein phosphatase PP2ARts1 leads to temperature and hyperosmotic stress sensitivity, yet the underlying mechanism and the scope of action of the phosphatase in the stress response remain elusive. Using a quantitative mass spectrometry-based approach we have identified a set of putative substrate proteins that show both hyperosmotic stress- and PP2ARts1-dependent changes in their phosphorylation pattern. A comparative analysis with published MS-shotgun data revealed that the phosphorylation status of many of these sites is regulated by the MAPKAP kinase Rck2, suggesting that the phosphatase antagonizes Rck2 signaling. Detailed gel mobility shift assays and protein-protein interaction analysis strongly indicate that Rck2 activity is directly regulated by PP2ARts1 via a SLiM B56-family interaction motif, revealing how PP2ARts1 influences the response to hyperosmotic stress in Yeast.

Polarized localization of yeast Pbs2 depends on osmostress, the membrane protein Sho1 and Cdc42.

In Saccharomyces cerevisiae cells, high external osmolarity leads to the activation of a p38-related mitogen-activated protein (MAP) kinase though Pbs2. Pbs2 tagged with green fluorescent protein (Pbs2-GFP) is evenly distributed in the cytoplasm but excluded from the nucleus before and after exposure to stress. Here we show that a catalytically inactive form of Pbs2 attains a highly polarised localization during osmostress. This phenomenon depends of the osmosensor Sho1 and on a functional Cdc42 GTPase. Cdc42, but not the actin cytoskeleton, influences Sho1-dependent activation of the MAP kinase. Sho1 itself accumulates at sites of polar growth, but independently of stress conditions and Cdc42. These observations allow us to define the sequence of events that occurs during propogation of osmostress signals.

Characterization of a gene product (Sec53p) required for protein assembly in the yeast endoplasmic reticulum.

SEC53, a gene that is required for completion of assembly of proteins in the endoplasmic reticulum in yeast, has been cloned, sequenced, and the product localized by cell fractionation. Complementation of a sec53 mutation is achieved with unique plasmids from genomic or cDNA expression banks. These inserts contain the authentic gene, a cloned copy of which integrates at the sec53 locus. An open reading frame in the insert predicts a 29-kD protein with no significant hydrophobic character. This prediction is confirmed by detection of a 28-kD protein overproduced in cells that carry SEC53 on a multicopy plasmid. To follow Sec53p more directly, a LacZ-SEC53 gene fusion has been constructed which allows the isolation of a hybrid protein for use in production of antibody. With such an antibody, quantitative immune decoration has shown that the sec53-6 mutation decreases the level of Sec53p at 37 degrees C, while levels comparable to wild-type are seen at 24 degrees C. An eightfold overproduction of Sec53p accompanies transformation of cells with a multicopy plasmid containing SEC53. Cell fractionation, performed with conditions that preserve the lumenal content of the endoplasmic reticulum (ER), shows Sec53p highly enriched in the cytosol fraction. We suggest that Sec53p acts indirectly to facilitate assembly in the ER, possibly by interacting with a stable ER component, or by providing a small molecule, other than an oligosaccharide precursor, necessary for the assembly event.

Regulation of meiotic S phase by Ime2 and a Clb5,6-associated kinase in Saccharomyces cerevisiae.

Cyclin-dependent kinase (Cdk) mutations that prevent entry into the mitotic cell cycle of budding yeast fail to block meiotic DNA replication, suggesting there may be fundamental differences between these pathways. However, S phase in meiosis was found to depend on the same B-type cyclins (Clb5 and Clb6) as it does in mitosis. Meiosis differs instead in the mechanism that controls removal of the Cdk inhibitor Sic1. Destruction of Sic1 and activation of a Clb5-dependent kinase in meiotic cells required the action of the meiosis-specific protein kinase Ime2, thereby coupling early meiotic gene expression to control of DNA replication for meiosis.

Sex, stress and integrity: the importance of MAP kinases in yeast.

To coordinate responses to environmental and cell autonomous signals, Saccharomyces cerevisiae utilizes distinct MAP kinase dependent signal transduction pathways. This offers the opportunity to compare the activation and attenuation mechanisms of MAP kinases in a single organism, and raises the issue of how the specificity of the individual signal pathways is maintained. Although many recent advances in our understanding of these pathways are due to biochemical reconstitution experiments, the most surprising results and insights have come from genetic analyses.

The SRF and MCM1 transcription factors.

The mammalian transcription factor SRF (serum-response factor) and the related Saccharomyces cerevisiae transcription factor MCM1 are the prototypes of a new class of dimeric DNA-binding proteins. Their function is regulated in part by the interactions of their DNA-binding domains with accessory proteins. Recent work has advanced the functional characterization of the contributions of SRF and MCM1, and their accessory proteins to transcriptional activation.

Mcm1 is required to coordinate G2-specific transcription in Saccharomyces cerevisiae.

In the budding yeast Saccharomyces cerevisiae, MCM1 encodes an essential DNA-binding protein that regulates transcription of many genes in cooperation with different associated factors. With the help of a conditional expression system, we show that Mcm1 depletion has a distinct effect on cell cycle progression by preventing cells from undergoing mitosis. Genes that normally exhibit a G2-to-M-phase-specific expression pattern, such as CLB1, CLB2, CDC5, SWI5, and ACE2, remain uninduced in the absence of functional Mcm1. In vivo footprinting experiments show that Mcm1, in conjunction with an Mcm1-recruited factor, binds to the promoter regions of SWI5 and CLB2 at sites shown to be involved in cell cycle regulation. However, promoter occupation at these sites is cell cycle independent, and therefore the regulatory system seems to operate on constitutively bound Mcm1 complexes. A gene fusion that provides Mcm1 with a strong transcriptional activation domain causes transcription of SWI5, CLB1, CLB2, and CDC5 at inappropriate times of the cell cycle. Thus, Mcm1 and a cooperating, cell cycle-regulated activation partner are directly involved in the coordinated expression of multiple G2-regulated genes. The arrest phenotype of Mcm1-depleted cells is consistent with low levels of Clb1 and Clb2 kinase. However, constitutive CLB2 expression does not suppress the mitotic defect, and therefore other essential activities required for the G2-to-M transition must also depend on Mcm1 function.

Pheromone-induced signal transduction in Saccharomyces cerevisiae requires the sequential function of three protein kinases.

Protein phosphorylation plays an important role in pheromone-induced differentiation processes of haploid yeast cells. Among the components necessary for signal transduction are the STE7 and STE11 kinases and either one of the redundant FUS3 and KSS1 kinases. FUS3 and presumably KSS1 are phosphorylated and activated during pheromone induction by a STE7-dependent mechanism. Pheromone also induces the accumulation of STE7 in a hyperphosphorylated form. This modification of STE7 requires the STE11 kinase, which is proposed to act before STE7 during signal transmission. Surprisingly, STE7 hyperphosphorylation also requires a functional FUS3 (or KSS1) kinase. Using in vitro assays for FUS3 phosphorylation, we show that pheromone activates STE7 even in the absence of FUS3 and KSS1. Therefore, STE7 activation must precede modification of FUS3 (and KSS1). These findings suggest that STE7 hyperphosphorylation is a consequence of its activation but not the determining event.

Pheromone-dependent G1 cell cycle arrest requires Far1 phosphorylation, but may not involve inhibition of Cdc28-Cln2 kinase, in vivo.

In yeast, the pheromone alpha-factor acts as an antiproliferative factor that induces G1 arrest and cellular differentiation. Previous data have indicated that Far1, a factor dedicated to pheromone-induced cell cycle arrest, is under positive and negative posttranslational regulation. Phosphorylation by the pheromone-stimulated mitogen-activated protein (MAP) kinase Fus3 has been thought to enhance the binding of Far1 to G1-specific cyclin-dependent kinase (Cdk) complexes, thereby inhibiting their catalytic activity. Cdk-dependent phosphorylation events were invoked to account for the high instability of Far1 outside early G1 phase. To confirm any functional role of Far1 phosphorylation, we undertook a systematic mutational analysis of potential MAP kinase and Cdk recognition motifs. Two putative phosphorylation sites that strongly affect Far1 behavior were identified. A change of serine 87 to alanine prevents the cell cycle-dependent degradation of Far1, causing enhanced sensitivity to pheromone. In contrast, threonine 306 seems to be an important recipient of an activating modification, as substitutions at this position abolish the G1 arrest function of Far1. Only the phosphorylated wild-type Far1 protein, not the T306-to-A substitution product, can be found in stable association with the Cdc28-Cln2 complex. Surprisingly, Far1-associated Cdc28-Cln2 complexes are at best moderately inhibited in immunoprecipitation kinase assays, suggesting unconventional inhibitory mechanisms of Far1.

PEP4 gene of Saccharomyces cerevisiae encodes proteinase A, a vacuolar enzyme required for processing of vacuolar precursors.

The proteinase A structural gene of Saccharomyces cerevisiae was cloned by using an immunological screening procedure that allows detection of yeast cells which are aberrantly secreting vacuolar proteins (J. H. Rothman, C. P. Hunter, L. A. Valls, and T. H. Stevens, Proc. Natl. Acad. Sci. USA, 83:3248-3252, 1986). A second cloned gene was obtained on a multicopy plasmid by complementation of a pep4-3 mutation. The nucleotide sequences of these two genes were determined independently and were found to be identical. The predicted amino acid sequence of the cloned gene suggests that proteinase A is synthesized as a 405-amino-acid precursor which is proteolytically converted to the 329-amino-acid mature enzyme. Proteinase A shows substantial homology to mammalian aspartyl proteases, such as pepsin, renin, and cathepsin D. The similarities may reflect not only analogous functions but also similar processing and intracellular targeting mechanisms for the two proteins. The cloned proteinase A structural gene, even when it is carried on a single-copy plasmid, complements the deficiency in several vacuolar hydrolase activities that is observed in a pep4 mutant. A strain carrying a deletion in the genomic copy of the gene fails to complement a pep4 mutant of the opposite mating type. Genetic linkage data demonstrate that integrated copies of the cloned proteinase A structural gene map to the PEP4 locus. Thus, the PEP4 gene encodes a vacuolar aspartyl protease, proteinase A, that is required for the in vivo processing of a number of vacuolar zymogens.

Osmotic stress-induced gene expression in Saccharomyces cerevisiae requires Msn1p and the novel nuclear factor Hot1p.

After a sudden shift to high osmolarity, Saccharomyces cerevisiae cells respond by transiently inducing the expression of stress-protective genes. Msn2p and Msn4p have been described as two transcription factors that determine the extent of this response. In msn2 msn4 mutants, however, many promoters still show a distinct rise in transcriptional activity upon osmotic stress. Here we describe two structurally related nuclear factors, Msn1p and a newly identified protein, Hot1p (for high-osmolarity-induced transcription), which are also involved in osmotic stress-induced transcription. hot1 single mutants are specifically compromised in the transient induction of GPD1 and GPP2, which encode enzymes involved in glycerol biosynthesis, and exhibit delayed glycerol accumulation after stress exposure. Similar to a gpd1 mutation, a hot1 defect can rescue cells from inappropriately high HOG pathway activity. In contrast, Hot1p has little influence on the osmotic stress induction of CTT1, where Msn1p appears to play a more prominent role. Cells lacking Msn1p, Msn2p, Msn4p, and Hot1p are almost devoid of the short-term transcriptional response of the genes GPD1, GPP2, CTT1, and HSP12 to osmotic stress. Such cells also show a distinct reduction in the nuclear residence of the mitogen-activated protein kinase Hog1p upon osmotic stress. Thus, Hot1p and Msn1p may define an additional tier of transcriptional regulators that control responses to high-osmolarity stress.

Kinase activity-dependent nuclear export opposes stress-induced nuclear accumulation and retention of Hog1 mitogen-activated protein kinase in the budding yeast Saccharomyces cerevisiae.

Budding yeast adjusts to increases in external osmolarity via a specific mitogen-activated protein kinase signal pathway, the high-osmolarity glycerol response (HOG) pathway. Studies with a functional Hog1-green fluorescent protein (GFP) fusion reveal that even under nonstress conditions the mitogen-activated protein kinase Hog1 cycles between cytoplasmic and nuclear compartments. The basal distribution of the protein seems independent of its activator, Pbs2, and independent of its phosphorylation status. Upon osmotic challenge, the Hog1-GFP fusion becomes rapidly concentrated in the nucleus from which it is reexported after return to an iso-osmotic environment or after adaptation to high osmolarity. The preconditions and kinetics of increased nuclear localization correlate with those found for the dual phosphorylation of Hog1-GFP. The duration of Hog1 nuclear residence is modulated by the presence of the general stress activators Msn2 and Msn4. Reexport of Hog1 to the cytoplasm does not require de novo protein synthesis but depends on Hog1 kinase activity. Thus, at least three different mechanisms contribute to the intracellular distribution pattern of Hog1: phosphorylation-dependent nuclear accumulation, retention by nuclear targets, and a kinase-induced export.

Nucleocytoplasmic traffic of MAP kinases.

MAPK pathways represent a unique extracellular signal response system. An important feature of such a multicomponent system appears to be the spatial intracellular organization of individual components. Recent studies demonstrate that the MAP kinases of such pathways are the molecular link between the plasma membrane sensors and the nuclear transcription factors. Stimulation of several MAPK pathways induces rapid and transient nuclear accumulation of MAP kinases. Investigations on the mode of regulation of this process using higher eukaryotes Erk2 and lower eukaryotes Hog1 and Sty1/Spc1 have revealed that at least three events contribute to signal-induced nuclear localization of these MAP kinases: activation by phosphorylation, regulated nuclear import and export, and nuclear retention.

Activation of the G2/M-specific gene CLB2 requires multiple cell cycle signals.

In budding yeast (Saccharomyces cerevisiae), the periodic expression of the G2/M-specific gene CLB2 depends on a DNA binding complex that mediates its repression during G1 and activation from the S phase to the exit of mitosis. The switch from low to high expression levels depends on the transcriptional activator Ndd1. We show that the inactivation of the Sin3 histone deacetylase complex bypasses the essential role of Ndd1 in cell cycle progression. Sin3 and its catalytic subunit Rpd3 associate with the CLB2 promoter during the G1 phase of the cell cycle. Both proteins dissociate from the promoter at the onset of the S phase and reassociate during G2 phase. Sin3 removal coincides with a transient increase in histone H4 acetylation followed by the expulsion of at least one nucleosome from the promoter region. Whereas the first step depends on Cdc28/Cln1 activity, Ndd1 function is required for the second step. Since the removal of Sin3 is independent of Ndd1 recruitment and Cdc28/Clb activity it represents a unique regulatory step which is distinct from transcriptional activation.

Identification, purification, and cloning of a polypeptide (PRTF/GRM) that binds to mating-specific promoter elements in yeast.

In yeast the alpha-specific regulators, alpha 1 and alpha 2 have been proposed to be DNA-binding proteins, both of which have to interact with an additional factor called PRTF or GRM, respectively, to exert their biological functions. Although the cis-acting sequence requirements for alpha 1 and alpha 2 are different, their target sequences share a common motif. PRTF or GRM is thought to act via this common DNA sequence; therefore, it has been suggested that they represent the same factor. I purified a protein that binds to this common promoter element by DNA affinity chromatography. The purified protein is able to recruit the alpha-specific activator alpha 1 to its binding sites, suggesting that it is indeed PRTF. Further evidence is presented to show that PRTF and GRM are the same protein and that PRTF plays a role in the activation of a-specific genes. Specific antibodies to the purified protein were obtained. They identify the protein as a component of DNA-protein complexes that formed with cell-type-specific promoter sequences. Using these antibodies, the gene encoding the protein was cloned from a yeast lambda gt11 expression library. The DNA sequence established that the gene encoding PRTF/GRM is identical with a previously described gene, FUN80 (essential factor of unknown function) or MCM1 (minichromosome maintenance). Sequence comparison showed further that PRTF/GRM shares similarities with a repressor from yeast, ARGRI, and the mammalian transcription factor SRF.

STE12, a protein involved in cell-type-specific transcription and signal transduction in yeast, is part of protein-DNA complexes.

The STE12 gene of Saccharomyces cerevisiae is essential for the expression of genes required for mating, such as those involved in pheromone response, and for genes unrelated to mating but regulated by the presence of an adjacent copy of the transposable element Ty1. We show that the STE12 protein is a component of specific DNA-protein complexes that form with transcriptional control elements from Ty1 and the alpha-pheromone receptor gene STE2. Although a sequence involved in pheromone-dependent transcriptional activation is protected in both complexes, competition experiments indicate that the complexes are intrinsically different from each other. We show that another factor involved in cell-type-specific transcription, PRTF/GRM, is a component of the complex with the STE2 fragment but not the Ty1 fragment. We propose that the STE12 product interacts with different transcription factors in different sequence contexts and that PRTF/GRM is one of these factors.

Overexpression of the G1-cyclin gene CLN2 represses the mating pathway in Saccharomyces cerevisiae at the level of the MEKK Ste11.

Basal and induced transcription of pheromone-dependent genes is regulated in a cell cycle-dependent way. FUS1, a gene strongly induced after pheromone treatment, shows high mRNA levels in mitosis and early G1 phase of the cell cycle, a decrease in G1 after START and again an increase in S phase. Overexpression of CLN2 was shown to repress the transcript number of pheromone-dependent genes (1). We asked whether the activities of components of the mating pathway fluctuate during the cell cycle. We were also interested in determining at what level Cln2 represses the signal transduction machinery. Here we show that the activity of the mitogen-activated protein kinase Fus3 indeed fluctuates during the cell cycle, reflecting the oscillations of the gene transcripts. CLN2 overexpression represses Fus3 kinase activity, independently of the phosphatase Msg5. Additionally, we show that the activity of the MEK Ste7 also fluctuates during the cell cycle. Increased Cln2 levels repress the ability of hyperactive STE11 alleles to induce the pathway. G protein-independent activation of Ste11 caused by an rga1 pbs2 mutation is resistant to high levels of Cln2 kinase. Therefore our results suggest that Cln2-dependent repression of the mating pathway occurs at the level of Ste11.

Cell cycle regulation of SW15 is required for mother-cell-specific HO transcription in yeast.

In haploid homothallic yeast, cell division gives rise to a mother cell that transiently transcribes the HO gene (as it undergoes START) and a daughter cell that does not. Consequently, only mother cells switch their mating types. Here, we test the proposition that a transcription factor called SWI5 is the "determinant" of mother-cell-specific HO transcription; that is, that SWI5 is the only factor missing in daughter cells. We show that SWI5 RNAs are cell-cycle regulated so that they are only produced after the post-START window of HO transcription. This regulation is vital for mother-cell specificity since constitutive transcription of SWI5 causes daughter cells to switch their mating types. We propose that SWI5 gene products are partitioned asymmetrically at cell division.