Hyperosmotic stress response and regulation of cell wall integrity in Saccharomyces cerevisiae share common functional aspects.

The osmosensitive phenotype of the hog1 strain is suppressed at elevated temperature. Here, we show that the same holds true for the other commonly used HOG pathway mutant strains pbs2 and sho1ssk2ssk22, but not for ste11ssk2ssk22. Instead, the ste11ssk2ssk2 strain displayed a hyperosmosensitive phenotype at 37 degrees C. This phenotype is suppressed by overexpression of LRE1, HLR1 and WSC3, all genes known to influence cell wall composition. The suppression of the temperature-induced hyperosmosensitivity by these genes prompted us to investigate the role of STE11 and other HOG pathway components in cellular integrity and, indeed, we were able show that HOG pathway mutants display sensitivity to cell wall-degrading enzymes. LRE1 and HLR1 were also shown to suppress the cell wall phenotypes associated with the HOG pathway mutants. In addition, the isolated multicopy suppressor genes suppress temperature-induced cell lysis phenotypes of PKC pathway mutants that could be an indication for shared targets of the PKC pathway and high-osmolarity response routes.

Candidate osmosensors from Candida utilis and Kluyveromyces lactis: structural and functional homology to the Sho1p putative osmosensor from Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, increases in external osmolarity evoke osmostress-induced signalling via the HOG MAP kinase pathway. One of the upstream components of this signal transduction route is the putative osmosensor, Sho1p. With the aim to elucidate the molecular basis of osmosensing in budding yeast, we have cloned SHO1 homologues from Candida utilis and Kluyveromyces lactis which allowed determination of conserved domains of Sho1p. Results obtained from sequence comparisons, confirmed the importance of the transmembrane domains and the SH3 domain for Sho1p function. The K. lactis and S. cerevisiae Sho1p show the highest degree of homology, the isoform from C. utilis is a shorter protein. SHO1 from C. utilis, however, did complement the osmosensitivity of the sho1ssk2ssk22 strain by restoring HOG pathway function, since Hog1p dual phosphorylation after high osmotic challenge was restored in this strain after transformation with a plasmid bearing this SHO1 homologue.

Arabidopsis thaliana and Saccharomyces cerevisiae NHX1 genes encode amiloride sensitive electroneutral Na+/H+ exchangers.

Sodium at high millimolar levels in the cytoplasm is toxic to plant and yeast cells. Sequestration of Na(+) ions into the vacuole is one mechanism to confer Na(+)-tolerance on these organisms. In the present study we provide direct evidence that the Arabidopsis thaliana At-NHX1 gene and the yeast NHX1 gene encode low-affinity electroneutral Na(+)/H(+) exchangers. We took advantage of the ability of heterologously expressed At-NHX1 to functionally complement the yeast nhx1-null mutant. Experiments on vacuolar vesicles isolated from yeast expressing At-NHX1 or NHX1 provided direct evidence for pH-gradient-energized Na(+) accumulation into the vacuole. A major difference between NHX1 and At-NHX1 is the presence of a cleavable N-terminal signal peptide (SP) in the former gene. Fusion of the SP to At-NHX1 resulted in an increase in the magnitude of Na(+)/H(+) exchange, indicating a role for the SP in protein targeting or regulation. Another distinguishing feature between the plant and yeast antiporters is their sensitivity to the diuretic compound amiloride. Whereas At-NHX1 was completely inhibited by amiloride, NHX1 activity was reduced by only 20-40%. These results show that yeast as a heterologous expression system provides a convenient model to analyse structural and regulatory features of plant Na(+)/H(+) antiporters.

The control of intracellular glycerol in Saccharomyces cerevisiae influences osmotic stress response and resistance to increased temperature.

Glycerol has been demonstrated to serve as the major osmolyte of Saccharomyces cerevisiae. Consistently, mutant strains gpd1gpd2 and gpp1gpp2, which are devoid of the main glycerol biosynthesis pathway, have been shown to be osmosensitive. In addition, the primary hyperosmotic stress response is affected in these strains. Hog1p phosphorylation turned out to be prolonged and osmostress-induced gene expression is delayed compared with the kinetics observed in wild-type cells. A hog1 deletion strain was previously found to contain lower internal glycerol and therefore displays an osmosensitive phenotype. Here, we show that the osmosensitivity of hog1 is suppressed by growth at 37 degrees C. We reasoned that this temperature-remedial osmoresistance might be caused by a higher intracellular glycerol level at the elevated temperature. This hypothesis was confirmed by measurement of the glycerol concentration, which was shown to be similar for wild type and hog1 cells only at elevated growth temperatures. In agreement with this finding, hog1 cells containing an fps1 allele, encoding a constitutively open glycerol channel, have lost their temperature-remedial osmoresistance. Furthermore, gpd1gpd2 and gpp1gpp2 strains were found to be temperature sensitive. The growth defect of these strains could be suppressed by adding external glycerol. In conclusion, the ability to control glycerol levels influences proper osmostress-induced signalling and the cellular potential to grow at elevated temperatures. These data point to an important, as yet unidentified, role of glycerol in cellular functioning.

Response of Saccharomyces cerevisiae to severe osmotic stress: evidence for a novel activation mechanism of the HOG MAP kinase pathway.

The HOG/p38 MAP kinase route is an important stress-activated signal transduction pathway that is well conserved among eukaryotes. Here, we describe a novel mechanism of activation of the HOG pathway in budding yeast. This mechanism operates upon severe osmostress conditions (1.4 M NaCl) and is independent of the Sln1p and Sho1p osmosensors. The alternative input feeds into the HOG pathway MAPKK Pbs2p and requires activation of Pbs2p by phosphorylation. We show that, upon severe osmotic shock, Hog1p nuclear accumulation and phosphorylation is delayed compared with mild stress. Moreover, both events lost their transient pattern, presumably because of the absence of negative feedback mediated by Ptp2p tyrosine phosphatase, which we found to be localized in the nucleus. Under severe osmotic stress conditions, the delayed nuclear accumulation correlates with a delay in stress-responsive gene expression. Severe osmoshock leads to a situation in which active and nuclear-localized Hog1p is transiently unable to induce transcription of osmotic stress-responsive genes. It also appeared from our studies that the Sho1p osmosensor is less active under severe osmotic stress conditions, whereas the Sln1p/Ypd1p/Ssk1p sensor and signal transducer functions normally under these circumstances.

Different roles for abf1p and a T-rich promoter element in nucleosome organization of the yeast RPS28A gene.

In vivo mutational analysis of the yeast RPS28A ribosomal protein (rp-)gene promoter demonstrated that both the Abf1p binding site and the adjacent T-rich element are essential for efficient transcription. In vivo Mnase and DNaseI digestion showed that the RPS28A promoter contains a 50-60 bp long nucleosome-free region directly downstream from the Abf1p binding site, followed by an ordered array of nucleosomes. Mutating either the Abf1p binding site or the T-rich element has dramatic, but different, effects on the local chromatin structure. Failure to bind Abf1p appears to cause nucleosome positioning to become disorganized as concluded from the complete disappearance of Mnase hypersensitive sites. On the other hand, mutation of the T-rich element causes the downstream nucleosomal array to shift by approximately 50 bp towards the Abf1p site, resulting in loss of the nucleosome-free region downstream of Abf1p. We conclude that Abf1p is a strong organizer of local chromatin structure that appears to act as a nucleosomal boundary factor requiring the downstream T-rich element to create a nucleosome-free region.

Lipoprotein cholesterol uptake mediates up-regulation of bile-acid synthesis by increasing cholesterol 7alpha-hydroxylase but not sterol 27-hydroxylase gene expression in cultured rat hepatocytes.

Lipoproteins may supply substrate for the formation of bile acids, and the amount of hepatic cholesterol can regulate bile-acid synthesis and increase cholesterol 7alpha-hydroxylase expression. However, the effect of lipoprotein cholesterol on sterol 27-hydroxylase expression and the role of different lipoproteins in regulating both enzymes are not well established. We studied the effect of different rabbit lipoproteins on cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase in cultured rat hepatocytes. beta-Migrating very-low-density lipoprotein (betaVLDL) and intermediate-density lipoprotein (IDL) caused a significant increase in the intracellular cholesteryl ester content of cells (2. 3- and 2-fold, respectively) at a concentration of 200 microgram of cholesterol/ml, whereas high-density lipoprotein (HDL, 50% v/v), containing no apolipoprotein E (apo E), showed no effect after a 24-h incubation. betaVLDL and IDL increased bile-acid synthesis (1. 9- and 1.6-fold, respectively) by up-regulation of cholesterol 7alpha-hydroxylase activity (1.7- and 1.5-fold, respectively). Dose- and time-dependent changes in cholesterol 7alpha-hydroxylase mRNA levels and gene expression underlie the increase in enzyme activity. Incubation of cells with HDL showed no effect. Sterol 27-hydroxylase gene expression was not affected by any of the lipoproteins added. Transient-expression experiments in hepatocytes, transfected with a promoter-reporter construct containing the proximal 348 nucleotides of the rat cholesterol 7alpha-hydroxylase promoter, showed an enhanced gene transcription (2-fold) with betaVLDL, indicating that a sequence important for a cholesterol-induced transcriptional response is located in this part of the cholesterol 7alpha-hydroxylase gene. The extent of stimulation of cholesterol 7alpha-hydroxylase is associated with the apo E content of the lipoprotein particle, which is important in the uptake of lipoprotein cholesterol. We conclude that physiological concentrations of cholesterol in apo E-containing lipoproteins increase bile-acid synthesis by stimulating cholesterol 7alpha-hydroxylase gene transcription, whereas HDL has no effect and sterol 27-hydroxylase is not affected.

DNA-binding requirements of the yeast protein Rap1p as selected in silico from ribosomal protein gene promoter sequences.

MOTIVATION: High-level transcriptional activation of most ribosomal protein (rp) genes in Saccharomyces cerevisiae is promoted by the global DNA-binding factor Rap1p. The creation of the complete database of yeast rp gene promoter sequences enabled us to develop a computational selection strategy aimed at acquiring detailed information concerning the DNA-binding specificity of Rap1p. RESULTS: Rap1p sites in rp gene promoters are often found in duplicate, exhibiting strong preferences in both spacing and orientation. Using these preferences, a weight matrix was selected that represents the in vivo binding requirements of Rap1p. The resulting matrix renders the identification of functional Rap1p binding sites more accurate and allowed us to re-evaluate previous in vitro data. Tandemly arranged Rap1p binding sites appear to be typical for rp gene promoters and differ in preferred spacing from sites occurring in (sub)telomeric repeats. The preferred spacing that is found in duplicate Rap1p binding sites of rp gene promoters is restricted to a small window between 15 and 26 bp. This is proposed to reflect the borders within which binding co-operativity operates. The data presented clearly illustrate that computational selection strategies provide information that reaches beyond experimental data. AVAILABILITY: The rp database is available at the url: http://www. chem.vu.nl/BMB/Database.html.

A search in the genome of Saccharomyces cerevisiae for genes regulated via stress response elements.

Stress response elements (STREs, core consensus AG4 or C4T) have been demonstrated previously to occur in the upstream region of a number of genes responsive to induction by a variety of stress signals. This stress response is mediated by the homologous transcription factors Msn2p and Msn4p, which bind specifically to STREs. Double mutants (msn2 msn4) deficient in these transcription factors have been shown to be hypersensitive to severe stress conditions. To obtain a more representative overview of the set of yeast genes controlled via this regulon, a computer search of the Saccharomyces cerevisiae genome was carried out for genes, which, similar to most known STRE-controlled genes, exhibit at least two STREs in their upstream region. In addition to the great majority of genes previously known to be controlled via STREs, 69 open reading-frames were detected. Expression patterns of a set of these were examined by grid filter hybridization, and 14 genes were examined by Northern analysis. Comparison of the expression patterns of these genes demonstrates that they are all STRE-controlled although their detailed expression patterns differ considerably.

The list of cytoplasmic ribosomal proteins of Saccharomyces cerevisiae.

Screening of the complete genome sequence from the yeast Saccharomyces cerevisiae has enabled us to compile a complete list of the genes encoding cytoplasmic ribosomal proteins in this organism. Putative ribosomal protein genes were selected primarily on the basis of the sequence similarity of their products with ribosomal proteins from other eukaryotic organisms, in particular the rat. These genes were subsequently screened for typical yeast rp-gene characteristics, viz. (1) a high codon adaptation index; (2) their promoter structure and (3) their responses to changes in growth conditions. The yeast genome appears to carry 78 different genes, of which 59 are duplicated, encoding 32 different small-subunit and 46 large-subunit proteins. A new nomenclature for these ribosomal proteins is proposed.

Transcriptional regulation of SUP35 and SUP45 in Saccharomyces cerevisiae.

SUP35 and SUP45 encode translational release factors in the yeast Saccharomyces cerevisiae. In addition, Sup35p is related to the cytoplasmically inherited prion-like phenotype [PSI+]. The vital cellular role of Sup35p and Sup45p prompted us to study the regulation of transcription of the corresponding genes. Since the [PSI] state of the yeast strain affects the abundance of Sup35p and Sup45p, both [PSI+] and [psi-] variants were included in these analyses. It turned out that SUP35 and SUP45 transcript levels are regulated by nutritional changes and stress in a way strikingly similar to those of ribosomal protein genes. The [PSI] state did not influence the respective transcript levels nor their regulation, although HSP12 (as a monitor of general stress-responsive) gene expression appeared to differ in the two variant strains. The transcription activation sites of SUP35 and SUP45 were mapped using deletion analysis of the respective promoter-reporter fusion genes. The UAS in both cases was found to consist of an Abf1p-site and a T-rich element. Also in this respect SUP35 and SUP45 show a notable resemblance with ribosomal protein genes. Evidence was found that SUP35 in addition harbors a potential internal promoter element which became active after progressive 5'-deletion removing the first of the three in-frame ATGs.

A new nomenclature for the cytoplasmic ribosomal proteins of Saccharomyces cerevisiae.

The availability of the complete sequence of the Saccharomyces cerevisiae genome has allowed a comprehensive analysis of the genes encoding cytoplasmic ribosomal proteins in this organism. On the basis of this complete inventory a new nomenclature for the yeast ribosomal proteins is presented.

Ribosomal protein gene transcription in Saccharomyces cerevisiae shows a biphasic response to nutritional changes.

Nutrients are major determinants of ribosomal protein (rp-) gene transcription in Saccharomyces cerevisiae. In order to investigate the molecular mechanisms underlying this nutritional control, yeast mutants that display defects in the glucose up-shift response of rp-gene transcription were isolated. Interestingly, although growth of these mutants on glucose-containing medium was severely affected an initial increase in rp-gene transcription by nutritional up-shift was still observed. However, at later time points, rp-mRNA levels decreased strongly. Various other types of severe growth limitation also did not prevent the initial up-shift in transcription. The results suggest that the glucose up-shift response of rp-gene transcription comprises two phases: an initial, transient response independent of the actual growth potential, and a sustained response which is dependent on growth and requires both glucose and adequate nitrogen sources. Previously, it was found that protein kinase A (Pka) mediates the initial up-shift response, without the need for regulation of Pka activity by cAMP. The present data substantiate that, besides the RAS/adenylate cyclase pathway, an alternative pathway through Pka regulates rp-gene transcription. In addition, evidence is presented that the sustained response does not require Pka activity. Based on these results, taken together, a model is proposed in which rp-gene transcription is dynamically regulated by multiple signal transduction pathways.

Glucose-triggered signalling in Saccharomyces cerevisiae: different requirements for sugar phosphorylation between cells grown on glucose and those grown on non-fermentable carbon sources.

Addition of glucose or fructose to cells of the yeast Saccharomyces cerevisiae grown on a nonfermentable carbon source triggers within a few minutes posttranslational activation of trehalase, repression of the CTT1 (catalase) and SSA3 (Hsp70) genes, and induction of the ribosomal protein genes RPL1, RPL25 and RPS33. By using appropriate sugar kinase mutants, it was shown that rapid glucose- or fructose-induced activation of trehalase requires phosphorylation of the sugar. On the other hand, partial induction of RPL1, RPL25 and RPS33 as well as partial repression of CTT1 and SSA3 were observed in the absence of sugar phosphorylation. In glucose-grown nitrogen-starved yeast cells readdition of a nitrogen source triggers activation of trehalase in a glucose- or fructose-dependent way, but with no apparent requirements for phosphorylation of the sugar. Repression of CTT1 and SSA3 under the same conditions was also largely dependent on the presence of the sugar and also in these cases there was a strong effect when the sugar could not be phosphorylated. Nitrogen induction of RPL1, RPL25 and RPS33 was much less dependent on the presence of the sugar, and only phosphorylated sugar caused a further increase in expression. These results show that two glucose-dependent signalling pathways, which can be distinguished on the basis of their requirement for glucose phosphorylation, appear to be involved in activation of trehalase, repression of CTT1 and SSA3 and induction of ribosomal protein genes. They also show that nutrient-induced repression of CTT1 and SSA3 is not a response to improvement of the growth conditions because the addition of nonmetabolizable sugar does not ameliorate the growth conditions. Similarly, the upshift in ribosomal protein synthesis cannot be a response to increased availability of energy or biosynthetic capacity derived from glucose, but it is apparently triggered to a significant extent by specific detection of glucose as such.

C-terminal domains of general regulatory factors Abf1p and Rap1p in Saccharomyces cerevisiae display functional similarity.

Abf1p and Rap1p are global regulatory factors which play an essential role in the transcription activation of yeast ribosomal protein genes. This functional link prompted us to investigate whether these factors may be functionally interchangeable. We focused on the indispensable C-terminal portions of both factors and performed mutual domain swaps. The functional capacity of the resulting hybrid proteins was subsequently examined using yeast strains conditionally expressing either the ABF1 or the RAP1 gene. Both the Abf1p-Rap1p and the Rap1p-Abf1p fusion proteins were found to be able to complement the growth defect of the respective strains. Furthermore, Abf1p and Rap1p are both able to promote transcription of a reporter gene through a combination of the respective binding site and a T-rich promoter element. These data strongly suggest that the C-terminal domains of Abf1p and Rap1p have, at least partially, identical functions. Finally, a deletion analysis of the so far largely uncharacterized C-terminal domain of Abf1p was performed, which revealed that two regions of 50 amino acids can perform all essential Abf1p functions.

The Saccharomyces cerevisiae HSP12 gene is activated by the high-osmolarity glycerol pathway and negatively regulated by protein kinase A.

The HSP12 gene encodes one of the two major small heat shock proteins of Saccharomyces cerevisiae. Hsp12 accumulates massively in yeast cells exposed to heat shock, osmostress, oxidative stress, and high concentrations of alcohol as well as in early-stationary-phase cells. We have cloned an extended 5'-flanking region of the HSP12 gene in order to identify cis-acting elements involved in regulation of this highly expressed stress gene. A detailed analysis of the HSP12 promoter region revealed that five repeats of the stress-responsive CCCCT motif (stress-responsive element [STRE]) are essential to confer wild-type induced levels on a reporter gene upon osmostress, heat shock, and entry into stationary phase. Disruption of the HOG1 and PBS2 genes leads to a dramatic decrease of the HSP12 inducibility in osmostressed cells, whereas overproduction of Hog1 produces a fivefold increase in wild-type induced levels upon a shift to a high salt concentration. On the other hand, mutations resulting in high protein kinase A (PKA) activity reduce or abolish the accumulation of the HSP12 mRNA in stressed cells. Conversely, mutants containing defective PKA catalytic subunits exhibit high basal levels of HSP12 mRNA. Taken together, these results suggest that HSP12 is a target of the high-osmolarity glycerol (HOG) response pathway under negative control of the Ras-PKA pathway. Furthermore, they confirm earlier observations that STRE-like sequences are responsive to a broad range of stresses and that the HOG and Ras-PKA pathways have antagonistic effects upon CCCCT-driven transcription.

Global regulators of ribosome biosynthesis in yeast.

Three abundant ubiquitous DNA-binding protein factors appear to play a major role in the control of ribosome biosynthesis in yeast. Two of these factors mediate the regulation of transcription of ribosomal protein genes (rp-genes) in yeasts. Most yeast rp-genes are under transcriptional control of Rap1p (repressor-activator protein), while a small subset of rp-genes is activated through Abf1p (ARS binding factor). The third protein, designated Reb1p (rRNA enhancer binding protein), which binds strongly to two sites located upstream of the enhancer and the promoter of the rRNA operon, respectively, appears to play a crucial role in the efficient transcription of the chromosomal rDNA. All three proteins, however, have many target sites on the yeast genome, in particular, in the upstream regions of several Pol II transcribed genes, suggesting that they play a much more general role than solely in the regulation of ribosome biosynthesis. Furthermore, some evidence has been obtained suggesting that these factors influence the chromatin structure and creat a nucleosome-free region surrounding their binding sites. Recent studies indicate that the proteins can functionally replace each other in various cases and that they act synergistically with adjacent additional DNA sequences. These data suggest that Abf1p, Rap1p, and Reb1p are primary DNA-binding proteins that serve to render adjacent cis-acting elements accessible to specific trans-acting factors.

Stress-induced transcriptional activation.

Living cells, both prokaryotic and eukaryotic, employ specific sensory and signalling systems to obtain and transmit information from their environment in order to adjust cellular metabolism, growth, and development to environmental alterations. Among external factors that trigger such molecular communications are nutrients, ions, drugs and other compounds, and physical parameters such as temperature and pressure. One could consider stress imposed on cells as any disturbance of the normal growth condition and even as any deviation from optimal growth circumstances. It may be worthwhile to distinguish specific and general stress circumstances. Reasoning from this angle, the extensively studied response to heat stress on the one hand is a specific response of cells challenged with supra-optimal temperatures. This response makes use of the sophisticated chaperoning mechanisms playing a role during normal protein folding and turnover. The response is aimed primarily at protection and repair of cellular components and partly at acquisition of heat tolerance. In addition, heat stress conditions induce a general response, in common with other metabolically adverse circumstances leading to physiological perturbations, such as oxidative stress or osmostress. Furthermore, it is obvious that limitation of essential nutrients, such as glucose or amino acids for yeasts, leads to such a metabolic response. The purpose of the general response may be to promote rapid recovery from the stressful condition and resumption of normal growth. This review focuses on the changes in gene expression that occur when cells are challenged by stress, with major emphasis on the transcription factors involved, their cognate promoter elements, and the modulation of their activity upon stress signal transduction. With respect to heat shock-induced changes, a wealth of information on both prokaryotic and eukaryotic organisms, including yeasts, is available. As far as the concept of the general (metabolic) stress response is concerned, major attention will be paid to Saccharomyces cerevisiae.

Transcription activation of yeast ribosomal protein genes requires additional elements apart from binding sites for Abf1p or Rap1p.

All ribosomal protein (rp) gene promoters from Saccharomyces cerevisiae studied so far contain either (usually two) binding sites for the global gene regulator Rap1p or one binding site for another global factor, Abf1p. Previous analysis of the rpS33 and rpL45 gene promoters suggested that apart from the Abf1 binding site, additional cis-acting elements play a part in transcription activation of these genes. We designed a promoter reconstruction system based on the beta-glucuronidase reporter gene to examine the role of the Abf1 binding site and other putative cis-acting elements in promoting transcription. An isolated Abf1 binding site turned out to be a weak activating element. A T-rich sequence derived from the rpS33 proximal promoter was found to be stronger, but full transcription activation was only achieved by a combination of these elements. Both in the natural rpL45 promoter and in the reconstituted promoter, a Rap1 binding site could functionally replace the Abf1 binding site. Characteristic rp gene nutritional control of transcription, evoked by a carbon source upshift or by nitrogen re-feeding to nitrogen starved cells, could only be mediated by the combined Abf1 (or Rap1) binding site and T-rich element and not by the individual elements. These results demonstrate that Abf1p and Rap1p do not activate rp genes in a prototypical fashion, but rather may serve to potentiate transcription activation through the T-rich element.