In Saccharomyces cerevisiae the subcellular distribution of Bcy1 is carbon source dependent. In glucose-grown cells, Bcy1 is almost exclusively nuclear, while it appears more evenly distributed between nucleus and cytoplasm in carbon source-derepressed cells. Here we show that phosphorylation of its N-terminal domain directs Bcy1 to the cytoplasm. Biochemical fractionation revealed that the cytoplasmic fraction contains mostly phosphorylated Bcy1, whereas unmodified Bcy1 is predominantly present in the nuclear fraction. Site-directed mutagenesis of two clusters (I and II) of serines near the N terminus to alanine resulted in an enhanced nuclear accumulation of Bcy1 in ethanol-grown cells. In contrast, substitutions to Asp led to a dramatic increase of cytoplasmic localization in glucose-grown cells. Bcy1 modification was found to be dependent on Yak1 kinase and, consequently, in ethanol-grown yak1 cells the Bcy1 remained nuclear. A two-hybrid screen aimed to isolate genes encoding proteins that interact with the Bcy1 N-terminal domain identified Zds1. In ethanol-grown zds1 cells, cytoplasmic localization of Bcy1 was largely absent, while overexpression of ZDS1 led to increased cytoplasmic Bcy1 localization. Zds1 does not regulate Bcy1 modification since this was found to be unaffected in zds1 cells. However, in zds1 cells cluster II-mediated, but not cluster I-mediated, cytoplasmic localization of Bcy1 was found to be absent. Altogether, these results suggest that Zds1-mediated cytoplasmic localization of Bcy1 is regulated by carbon source-dependent phosphorylation of cluster II serines, while cluster I acts in a Zds1-independent manner.Throughout the eukaryotic kingdom cyclic AMP (cAMP)-dependent protein kinases (PKAs) play important and diverse roles in signal transduction (for reviews, see references 2, 7, and 28 and references therein). Structurally, PKAs are conserved, consisting of two catalytic subunits that bind, in their inactive configuration, to a regulatory subunit homodimer. Binding of cAMP to the regulatory subunit results in dissociation, and thereby activation, of the catalytic subunits (7, 28). The multitude of intracellular PKA substrates and their different subcellular distribution raises important questions about the specificity, timing, and substrate targeting of PKA-mediated signaling. One regulatory level to ensure proper signal transduction is specific targeting of signaling components to subcellular compartments. In multicellular eukaryotes A-kinase anchor proteins (AKAPs) have been identified that target type I or type II (RI or RII) PKA-regulatory subunits to their effector substrates localized in various subcellular compartments (for recent reviews, see references 5 and 6 and references therein). AKAPs possess a site for constitutive avid binding of RI or RII and a targeting domain that complexes with subcellular structures. Directing PKA to specific microenvironments facilitates phosphorylation of colocalized effector molecules.In contrast to cells from multicellular organisms, yeast...

The existence of specific DL-glycerol-3-phosphatase (EC 3.1.3.21) activity in extracts of Saccharomyces cerevisiae was confirmed by examining strains lacking nonspecific acid and alkaline phosphatase activities. During purification of the glycerol-3-phosphatase, two isozymes having very similar molecular weights were isolated by gel filtration and anion exchange chromatography. By microsequencing of trypsin-generated peptides the corresponding genes were identified as previously sequenced open reading frames of unknown function. The two genes, GPP1 (YIL053W) and GPP2 (YER062C) encode proteins that show 95% amino acid identity and have molecular masses of 30.4 and 27.8 kDa, respectively. The intracellular concentration of Gpp2p increases in cells subjected to osmotic stress, while the production of Gpp1p is unaffected by changes of external osmolarity. Both isoforms have a high specificity for DL-glycerol-3-phosphate, pH optima at 6.5, and K m G3P in the range of 3-4 mM. The osmotic induction of Gpp2p is blocked in cells that are defective in the HOG-mitogenactivated protein kinase pathway, indicating that GPP2 is a target gene for this osmosensing signal transduction pathway. Together with DOG1 and DOG2, encoding two highly homologous enzymes that dephosphorylate 2-deoxyglucose-6-phosphate, GPP1 and GPP2 constitute a new family of genes for low molecular weight phosphatases.

Phenotypic differences between planktonic bacteria and those attached to abiotic surfaces exist, but the mechanisms involved in the adhesion response of bacteria are not well understood. By the use of twodimensional (2D) polyacrylamide gel electrophoresis, we have demonstrated that attachment of Escherichia coli to abiotic surfaces leads to alteration in the composition of outer membrane proteins. A major decrease in the abundance of resolved proteins was observed during adhesion of type 1-fimbriated E. coli strains, which was at least partly caused by proteolysis. Moreover, a study of fimbriated and nonfimbriated mutants revealed that these changes were due mainly to type 1 fimbria-mediated surface contact and that only a few changes occurred in the outer membranes of nonfimbriated mutant strains. Protein synthesis and proteolytic degradation were involved to different extents in adhesion of fimbriated and nonfimbriated cells. While protein synthesis appeared to affect adhesion of only the nonfimbriated strain, proteolytic activity mostly seemed to contribute to adhesion of the fimbriated strain. Using matrix-assisted laser desorption ionization-time of flight mass spectrometry, six of the proteins resolved by 2D analysis were identified as BtuB, EF-Tu, OmpA, OmpX, Slp, and TolC. While the first two proteins were unaffected by adhesion, the levels of the last four were moderately to strongly reduced. Based on the present results, it may be suggested that physical interactions between type 1 fimbriae and the surface are part of a surface-sensing mechanism in which protein turnover may contribute to the observed change in composition of outer membrane proteins. This change alters the surface characteristics of the cell envelope and may thus influence adhesion.

The salt-instigated protein expression of Saccharomyces cerevisiae during growth in either 0.7 or 1.4 M NaCl was studied by two-dimensional polyacrylamide gel electrophoresis. The 73 protein spots that were identified as more than 3-fold responsive in 1.4 M NaCl were further grouped by response class (halometric, low-salt, and high-salt regulation). Roughly 40% of these responsive proteins were found to decrease in expression, while at higher magnitudes of change (>8-fold) only induction was recorded. Enolase 1 (Eno1p) was the most increasing protein by absolute numbers per cell, but not by -fold change, and the enzymes involved in glycerol synthesis, Gpd1p and Gpp2p, were also induced to a similar degree as Eno1p. We furthermore present evidence for salt induction of glycerol dissimilation via dihydroxyacetone and also identify genes putatively encoding the two enzymes involved; dihydroxyacetone kinase (DAK1 and DAK2) and glycerol dehydrogenase (YPR1 and GCY1). The GPD1, GPP2, GCY1, DAK1, and ENO1 genes all displayed a halometric increase in the amount of transcript. This increase was closely linked to the salt-induced rate of protein synthesis of the corresponding proteins, indicating mainly transcriptional regulation of expression for these genes. A consensus element with homology to the URS sequence of the ENO1 promoter was found in the promoters of the GPD1, GPP2, GCY1, and DAK1 genes.

Sulfur metabolism has been studied extensively and is, to a large degree, universal for eukaryotes [1][2][3][4]. However, there are still predicted pathways in which all enzymes ⁄ genes have not been identified. Sulfur is needed in the form of the amino acids cysteine and methionine, as well as S-adenosylmethionine, the adenosylated form of methionine. S-adenosylmethionine has many different functions in the cell, including as a starting point for the synthesis of polyamines: putrescine, spermine and spermidine. Polyamines are important for growth, and yeast has an absolute requirement for putrescine and spermidine, as studied in deletion mutants of biosynthetic enzymes [5,6]. During the formation of spermidine and spermine, the metabolite 5¢-methylthioadenosine (MTA) is formed, which contains the sulfur atom of methionine.As the assimilation of sulfur is strongly energy consuming in the form of redox equivalents [2], most organisms from bacteria to mammals and plants have evolved recycling pathways to reuse sulfur and regenerate methionine: the 'methionine salvage pathway' (MTA cycle) [7][8][9][10][11]. The cycle of mammals and yeast consists of six enzymatic steps and one spontaneous reaction. The active enzymes are MTA phosphorylase The methionine salvage pathway is universally used to regenerate methionine from 5¢-methylthioadenosine, a byproduct of certain reactions involving S-adenosylmethionine. We identified and verified the genes encoding the enzymes of all steps in this cycle in a commonly used eukaryotic model system: the yeast Saccharomyces cerevisiae. The genes encoding 5¢-methylthioribose-1-phosphate isomerase and 5¢-methylthioribulose-1-phosphate dehydratase are herein named MRI1 and MDE1, respectively. The 5¢-methylthioadenosine phosphorylase was verified as Meu1p, the 2,3-dioxomethiopentane-1-phosphate enolase ⁄ phosphatase as Utr4p and the aci-reductone dioxygenase as Adi1p. The homologue of the enolase ⁄ phosphatase gene, YNL010w, was excluded from its candidate role in the cycle. The methodology used involved auxotrophic growth tests and analysis of intracellular 5¢-methylthioadenosine in deletion mutants. The last step, a transamination of 4-methylthio-2-oxobutyrate to yield methionine, was found to be a highly redundant step. It was catalysed by amino acid transaminases, mainly coupled with aromatic and branched chain amino acids as amino donors, but also with proline, lysine and glutamate ⁄ glutamine. The aromatic amino acid transaminases, Aro8p and Aro9p, and the branched chain amino acid transaminases, Bat1p and Bat2p, seemed to be the main enzymes exhibiting 4-methylthio-2-oxobutyrate transaminase activity. Bat2p was found to be less specific and used proline, lysine, tyrosine and glutamate as amino donors in addition to the branched chain amino acids. Thus, for the first time, all enzymes of the methionine salvage pathway were identified in a eukaryote.Abbreviations MOB, 4-methylthio-2-oxobutyrate; MTA, 5¢-methylthioadenosine; SGD, Saccharomyces Genome Database

The genes YML070W/DAK1 and YFL053W/DAK2 in the yeast Saccharomyces cerevisiae were characterized by a combined genetic and biochemical approach that firmly functionally classified their encoded proteins as dihydroxyacetone kinases (DAKs), an enzyme present in most organisms. The kinetic properties of the two isoforms were similar, exhibiting K m(DHA) of 22 and 5 M and K m(ATP) of 0.5 and 0.1 mM for Dak1p and Dak2p, respectively. We furthermore show that their substrate, dihydroxyacetone (DHA), is toxic to yeast cells and that the detoxification is dependent on functional DAK. The importance of DAK was clearly apparent for cells where both isogenes were deleted (dak1⌬dak2⌬), since this strain was highly sensitive to DHA. In the opposite case, overexpression of either DAK1 or DAK2 made the dak1⌬dak2⌬ highly resistant to DHA. In fact, overexpression of either DAK provided cells with the capacity to grow efficiently on DHA as the only carbon and energy source, with a generation time of about 5 h. The DHA toxicity was shown to be strongly dependent on the carbon and energy source utilized, since glucose efficiently suppresses the lethality, whereas galactose or ethanol did so to a much lesser extent. However, this suppression was found not to be explained by differences in DHA uptake, since uptake kinetics revealed a simple diffusion mechanism with similar capacity independent of carbon source. Salt addition strongly aggravated the DHA toxicity, independent of carbon source. Furthermore, the DHA toxicity was not linked to the presence of oxygen or to the known harmful agents methylglyoxal and formaldehyde. It is proposed that detoxification of DHA may be a vital part of the physiological response during diverse stress conditions in many species.

An interlaboratory comparison was conducted on the positional and quantitative reproducibility of yeast proteins resolved by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) using isoelectric focusing with immobilized pH gradient (pH 4-7) in the first dimension. The basic experimental set-up was as follows: one laboratory prepared and distributed a [35S]methionine-labeled total yeast protein extract (Göteborg, Sweden), another laboratory prepared the IPG strips to be used by all labs in this study (Munich, Germany), the third laboratory (Aarhus, Denmark) circulated the protocols and coordinated the modest attempts to unify them. Samples were run horizontally in the first dimension and vertically in the second. The gels were sent to Göteborg for processing by phosphoimager technology and computerized image analysis (PDQuest), and the 2-D PAGE resolved proteins were located and quantified automatically. A subset of 470 spots was manually matched in all gels out of an average of 1328 resolved proteins. The positional interlaboratory comparison revealed great pattern reproducibility, the correlation coefficient in no case being less than 0.9994. In absolute terms an average deviation of 2.8 mm (x-position) and 1.8 mm (y-position) were obtained for all nine gels (three gels per lab). The interlaboratory comparison of protein quantitation displayed higher variability, and the best correlation coefficient generated was 0.975. An average standard deviation of 34.5% was calculated for protein quantitation including all three labs, a value slightly higher than the intralaboratory variation (range 20-28%). Thus, despite differences in protocols, chemicals and equipment, the immobilized pH gradient technology gave extremely high positional and quantitative reproducibility. This will greatly facilitate the exchange of data and the establishment of multi-user image-based 2-D gel databases.

The influence of cAMP‐dependent protein kinase (PKA) on protein expression during exponential growth under osmotic stress was studied by two‐dimensional polyacrylamide gel electrophoresis (2D–PAGE). The responses of isogenic strains (tpk2Δtpk3Δ) with either constitutively low (tpk1w1), regulated (TPK1) or constitutively high (TPK1bcy1Δ) PKA activity were compared. The activity of cAMP‐dependent protein kinase (PKA) was shown to be a major determinant of osmotic shock tolerance. Proteins with increased expression during growth under sodium chloride stress could be grouped into three classes with respect to PKA activity, with the glycerol metabolic proteins GPD1, GPP2 and DAK1 standing out as independent of PKA. The other osmotically induced proteins displayed a variable dependence on PKA activity; fully PKA‐dependent genes were TPS1 and GCY1, partly PKA‐dependent genes were ENO1, TDH1, ALD3 and CTT1. The proteins repressed by osmotic stress also fell into distinct classes of PKA‐dependency. Ymr116c was PKA‐independent, while Pgi1p, Sam1p, Gdh1p and Vma1p were fully PKA‐dependent. Hxk2p, Pdc1p, Ssb1p, Met6p, Atp2p and Hsp60p displayed a partially PKA‐dependent repression. The promotors of all induced PKA‐dependent genes have STRE sites in their promotors suggestive of a mechanism acting via Msn2/4p. The mechanisms governing the expression of the other classes are unknown. From the protein expression data we conclude that a low PKA activity causes a protein expression resembling that of osmotically stressed cells, and furthermore makes cells tolerant to this type of stress. Copyright © 2000 John Wiley & Sons, Ltd.