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ELSEVIER FEMS Microbiology Letters 137 (1996) 1-8 Protein expression during exponential growth in 0.7 M NaCl medium of Saccharomyces cerevisiae Joakim Norbeck, Anders Blomberg * Department of General and Marine Microbiology, Lundberg Laboratory, Uniuersio of Gb;teborg, Medicinaregatan 9C, 413 90 Giiteborg, Sweden Received 1 December 1995; revised 22 December 1995; accepted 22 December 1995 Abstract Saccharomyces cereuisiae exponentially growing in basic or 0.7 M NaCl medium were isotopically labelled with 35S-methionine, followed by protein separation and quantification by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) combined with computerised image analysis. The electrophoretic separation resolved about 650 proteins of which 13 displayed significant and at least 2-fold changes in rate of synthesis during saline growth. By sequencing of 2D-PAGE resolved proteins, one of the 8 induced spot, p42.9/5.5, was shown to correspond to the full length (containing the N-terminal extension) product of the GPDI gene encoding the cytoplasmic glycerol 3-phosphate dehydrogenase. The expression of the TDH3 gene, glyceraldehyde 3-phosphate dehydrogenase, and the EN02 gene, enolase, decreased during growth in NaCl medium, declines hypothesised to have an impact on the flux to glycerol. Keywords: Saccharomyces cereuisiae; NaCl stress; Protein synthesis; 2D-PAGE; Protein sequencing; 1. Introduction The adaptive response of Saccharomyces cereto NaCl involves exclusion of Na+ to maintain low intracellular concentrations of this ion and an osmotic potential compensatory accumulation of the three-carbon polyol glycerol [I]. A key protein in the osmoregulatory response is the cytoplasmic enzyme glycerol 3-phosphate dehydrogenase (GPD) [2]. The activity and amount of GPD in cellular extracts is increased in response to high extracellular concentrations of NaCl [3], a regulation reportedly at the transcriptional level [4-71. uisiae * Corresponding author. Tel: +46 (31) 773 2589; fax: (31) 773 2599; E-mail: [email protected] 0378-1097/96/$15.00 0 1996 Federation PIZ SO378-1097(96)00006-7 of European +46 Microbiological GPDl; Glycolytic enzymes Computer aided analysis of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) generated images of isotopically labelled protein extracts have been performed in a quantitative investigation of the cellular adaptation process to NaCl containing medium [8]. It was reported that drastic expression changes occurred during the osmotic adaptation, with 138 proteins displaying a significant and at least 2-fold change in relative synthetic rate (induction or repression). The 20 highly responsive proteins with more than 8-fold changes in synthesis exhibited mainly increased rate of production; only two proteins were repressed. Responsive proteins during this one hour osmotic adaptation could be classified into 14 different regulatory families by their temporal response, revealing some of the intricate complexity Societies. All rights reserved of the transition physiology in S. cerer~isiur. lncreased glycerol accumulation seems to be one of the main osmoregulatory mechanisms even for actively proliferating cells, however, indications of a substantially altered metabolism by salt stress has also been presented [9]. Thus. a global study on the changes in protein synthesis during exponential growth in NaCl media was conducted in order to search for protein responders indicating additional salt-instigated cellular adjustments. 2. Materials and methods acetic acid 10% [v/v]>. The fixed gels were rinsed in milliQ for at least 2 h in order to attain their maximum size (roughly 25 X 25 cm>, before being dried on chromatography paper in a gel drier. The dried gels were exposed to Kodak X-AR film, exposure time depending on the amount of incorporated “Smethionine in the extracts. Gels corresponding to cells from 0 M NaCl were exposed for 1. 3 and 7 days, those from 0.7 M NaCl for 3, 7 and 30 days. Films were developed using a AGFA-CURIX 60 automatic developer. For each exposure time a calibration strip with a wide span of radioactivity was included [ 111. 2.1. Strain and mediu 2.5. Scanning and computevised Saccharomyes cerer,isiae strain Y41 (ATCC 38531) was maintained, grown and labelled in a defined minimal medium (YNB) with 0.5% (w/v) glucose, as reported earlier [IO]. The analysis was carried out using the PDQuest program v. 4. I (PDI, Huntington Station, New York) as earlier described [S]. Protein quantity was expressed as DPM in the spot relative the total amount of radioactivity loaded onto the first dimensional gels. 2.2. Label&g and sample preparation Labelling and sample preparation was performed in triplicates with cells grown in 10 ml of YNB medium at 3o”C, as described earlier [ 101. lndependent of growth medium a total amount of protein in the range 2.9-4.3 pg (average 3.4 pg) per ~1 extract was obtained. 2.3. 2D-PAGE electrophoresis The 2D-PAGE gels were run essentially as earlier reported [lo], with the following minor modifications: i> the second dimensional SDS-PAGE slab gels were 10% acrylamide [0.80/G bis] and ii) the effect was held constant at 1400 mW/gel and the gels were run overnight at 20°C. A maximum of 20 pg of total protein was loaded onto the first dimensional tubes and this amount of protein corresponded to approximately I .4 X IO6 DPM and 0.25 X 1Oh DPM for the control and the salt grown cells, respectively. 2.4. Spot risualisution l-2 After the completed run the gels were fixed for h in fixing solution (ethanol 50% [v/v] and 2.6. Quantification duta analysis of dominant spots Some of the dominant spots were saturated or distorted in shape and could not be accurately quantified by computerised image analysis. These spots were cut out, placed in 5 ml of Aquassure and counted in a scintillator. The spot corresponding to actin was included as a reference through which the quantifications of the dominant spots could be related to the spots quantified by image analysis. 2.7. Preparutirle 2D-PAGE and sequencing The preparative IEF gels were run as earlier described [lo]. Identification of the spots were performed by sequencing of reversed phase-HPLC fractionated peptides generated by trypsin digestion in-gel of 2D-PAGE resolved proteins [ 101. For the sequencing of Fbalp and Eno2p, proteins were visualised by copper staining [ 121. The protein spots were electroblotted to an immobilon-psq membrane (Millipore Inc.), using a Mini Trans-blotTM assembly (Bio-Rad), and sent to KEBO laboratories for N-terminal sequencing on an Applied biosystems (model 473A) protein sequencer. J. Norbeck, A. Biomberg / FEMS Microbiology Letters 137 (19%) 1-8 2.8. Measurements of activities of glycolytic enzymes The enzyme activity measurements were performed essentially as described previously [13]. The protein extraction method however differed slightly. 1.5 g of acid washed glass beads was added to a pellet of approximately 2.5 X lo9 cells together with protease inhibitors (Pefabloc @ 1 mg/ml, Leupeptin 3 pg/ml, Pepstatin 15 pg/ml and Bestatin 40 pg/ml) in 1 ml of buffer (Triethanolamine 10 mM, DTT 1 mM and EDTA-Na,, pH 7.5). After vortexing 4 X 30 s (with placement on ice > 1 min inbetween) the glass beads and debris were pelleted at 2000 X g for 3 min in a table top centrifuge. The supematant was further centrifuged at 18 000 X g for 15 min and the supematant from this step was used for all enzyme measurements. Care was taken to keep samples ice-cold at all times. 3. Results and discussion 3.1. Salinity induced changes in global protein synthesis Early respiratory phase cultures of strain Y41 of Saccharomyces cereuisiae, growing in non-saline medium, were used as inoculum into medium without NaCl (control) or to medium supplemented with 0.7 M NaCl. After a lag phase of roughly the same length in the two media (1.5 h) growth resumed, however, at a slower rate for the salinity stressed culture; the generation times were 2.2 and 3.2 h, respectively. Protein extracts were prepared from 35S-methionine labelled mid-exponential cultures, and were subjected to 2D-PAGE analysis. Irrespective of growth condition roughly 650 spots were detected and quantified in the gels. After manual matching of 261 spots in the different gels (leaving out spots which by visual inspection clearly not changed in rate of synthesis), computer comparisons of significant and more than 2-fold quantitative differences were allocated. Only proteins which exhibited a production level of at least 100 ppm (0.01%) in either growth condition are reported (Table 1, Fig. 1). The most salinity induced protein, p23.9/5.7, changed expression by a factor of 4.5. This protein was also the most highly expressed of all the respon- Table 3 1 Proteins exhibiting significant (by log t-test criteria) and at least 2-fold changes in synthesis (induction or repression) in NaCl grown S. cerecisiae M, /PI 0 M NaCl &Da/PHI (ppm) a 0.7 M NaCl n-fold change (ppm) Induced proteins 23.9/5.7 134 25.6/6.4 87 34.9/5.3 74 42.9/5.5 h 32 35.1/6.6 60.3 48.5/6.2 63 47 25.6/6.6 5 I .4/6.0 64 +_3l f 7 +54 532 *4l +I0 +19 f 9 602+16 380&- 19 286+ I7 113+27 149+ 10 153f 12 106f31 129+25 4.5 4.4 3.8 3.6 2.5 2.4 2.3 2.0 Repressed proteins 59.6/6.5 157 36.6/5.2 165 27.6/5.4 211 59.4/5.5 114 53.2/6.2 122 k27 k28 i25 +25 +20 15+28 47+65 86k20 52f26 61+23 10.7 3.5 2.4 2.2 2.0 Only proteins with an expression level of at least 100 ppm in the induced state have been included. Data represent mean of triplicate independent experiments + SD. a Relative rate of synthesis as parts per million. DPM recorded in the spot is related to the total amount of DPM loaded onto the first dimensional gel. b Identified as the product of the GPDI gene by microsequencing (Table 2). ZD-PAGE position indicated in Fig. 2. sive ones, and exhibited during exponential growth in 0.7 M NaCl an expression level of about 600 ppm. The small number of salt-responsive proteins and their minor expression change scored in this study for proliferating cells strongly contrasts the earlier reported cellular response during adaptation to salt, which was accompanied by a massive change in global protein synthesis; e.g. 18 proteins being induced at least eight fold [8]. A prominent feature of these highly NaCl-responsive proteins for adapting cells were their transient response, all exhibiting maximal rate of synthesis in the middle of the adaptation period. The data presented here further supports this transient expression, since few proteins exhibited induced levels during saline growth, and those that did so only increased their rate of synthesis to a minor extent. The protein that displayed the most dramatic change in expression was p59.6/6.5 which was repressed by a factor of about 10 to an expression level in the NaCl medium of only 15 ppm (Table 1). Thus, the transition physiology of yeast cells display great plasticity in terms of protein synthesis, probably responding to signals triggered by the initial osmotic dehydration [8]. Exponentially growing cells, on the other hand, has adapted to the new conditions and potentially re-established almost norma1 signalling levels, indicated by their less dramatic expression changes. 3.2. Protein ident$cation b!t tnicrosequet~cit~g Protein p42.9/5.5 (Fig. 1). exhibiting a 3.6-fold salt induction (Table I ). displayed M, and p I values similar to Gpdlp (theoretical value 42.8/5.3). Link- age of this NaCl responsive spot to the GPDl gene was unequivocally confirmed by sequencing of trypsin generated peptides (Table 2), and the obtained amino acid sequences clearly distinguished it from its recently cloned isogene product, Gpd2p [6]. It was also clear that the 30 amino acid N-terminal extension, previously believed to be cleaved off. was present in this salt responsive Gpdlp spot, as one of the trypsin generated peptides spanned residues 8- 16. This is interesting since the purified GPD enzyme [14] was devoid of the 30 most N-terminal amino acids and the sequence started at position 3 1 [ 151. This N-terminal discrepancy could be an outcome of differences in the physiological states of the cells at harvest (exponential versus stationary phase cultures). 70 50 30 610 PI (PW Fig. I. 2D-PAGE analysis of [ “S]methionine labelled proteins fol- salinity instigated protein expression changes in S. cerer~i.siw. The central portion of the gels (autoradiogram) is displayed, covering a pl range of 4.9-6.5 and a M, range of 24 to 100 kDa. (A) Cells growing exponentially in 0 M NaCl medium. (B) Cells growing exponentially in 0.7 M NaCl medium. Protein spots that exhibited a significant and more than 2-fold expression change in saline cultures compared to the control are indicated. Arrows mark position of spots with increased synthesis in 0.7 M NaCl medium and squares indicate spots with decreased synthesis in 0.7 M NaCl medium. Displayed are the raw scan of the longest exposures. J. Norbeck, A. Blomberg/ FEMS Microbiology Letters 137 (1996) 1-8 tentatively indicating novel intricate regulation of the Gpdl protein. The other salt-responsive spots were in our preparative gels not found at their predicted amounts (Table l), a discrepancy potentially caused by differential methionine contents. However, a number of other proteins in the 2D pattern of S. cereuisiue were identified by the sequencing procedures (Table 2) and these together with some of our previous identifications are depicted in Fig. 2. An identified protein that displayed an almost 2-fold change in expression during NaCl growth was Samlp, which was repressed 1.8 fold in salt (629 to 352 ppm). Samlp catalyses the synthesis of Sadenosylmethionine (AdoMet) from methionine and 5 ATP. AdoMet is an important methyl donor for transmethylation and is also the propylamino donor of polyamin biosynthesis [ 161. The Samlp down regulation in salt indicates somewhat decreased activities in these processes. In this context it is of interest that salt dependent differential regulation of the different SAM isogenes have been observed in plants, hypothesised to reflect NaCl imposed changes in cell wall synthesis [17]. The Pglucanase Bgl2p involved in cell wall synthesis in yeast was expressed to the same level in salt grown or control cultures. The biosynthetic proteins Met6p and Lys9p displayed salt invariant expression, indicating minor alterations by NaCl treatment to amino acid biosyn- 610 PI (PHI Fig. 2. Identified proteins in the ZD-PAGE pattern of S. cereuisiae. All spots indicated are identified by microsequencing, and sequences are reported in Table 2 or in [lo]. The following gene designations are used: ACTl, actin; ATP2, F, -subunit of the mitochondrial ATPase; ADHI, alcohol dehydrogenase I; BGL2, pglucanase; EFBl, translational elongation factor 1 p; EN01 and EN02, enolase A and Ef; FBAl, fructose bisphosphate dehydrogenase; GDHl, glutamate dehydrogenase (NADP+); GPDl, glycerol-3-phosphate dehydrogenase; HXK2, hexokinase PII; LYS9, saccharopine dehydrogenase; MET6, methionine synthetase; MET17, O-acetyl (homolserine sulthydrylase; PDCl, pyruvate decarboxylase; IPPI, inorganic pyrophosphatase; SAMl, S-adenosylmethionine synthetase I; SSA1/2, SSBl, SSB2 and SSEl, HSP 70 isogenes; TDH3 and TDH2, glyceraldehyde 3-phosphate dehydrogenase. For those who prefer the other orientation of 2D yeast patterns (acidic side to the right) the flipped version of this image is available at our WWW-2DPAGE database server: http://yeastZDPAGE.gmm.gu.se. 6 .I. Norbeck. A. Blomherg/ Table 2 Experimentally obtained protein or peptide sequence:, 2D-PAGE resolved proteins (listed in alphabetic order) M, /pI 40.0/5.5 46.1/5.3 28.3/-.29.5/-.42.8/6.2 35.8,‘6.1 49.0/5.6 42.9/5.5 Residues in Gene protein assigned AGFAXDD XIXELGIYP LLDAXVVCQ IGELAFNL IXESTVAGFL (QJLNASLADK WFNHIA AVSKVYARSVYDSRGNPTVE GVEQILKRKTGVIVGEDVHN FHPXVNLXIL EIGYLFGAY VIELGGTVVXL LNLTXXHLN 19- 25 371-379 390-398 24- 31 111-120 13- 21 50- 55 2- 21 h 2- 20h 79- 88 157-165 236-246 x- 16 160-173 45.4/5.2 ILXGFAWLGLF. FDXNYH(Y)VRP AXTYF(D)EQSN NYPNHI(I)L FVEGDNPEEF DLPNAD(K)ETDPF GIDLTNVXLPD DXXLAW 314-324 122-131 234-243 692-699 130-139 350-361 239-249 157-162 IDXVSSAQH 187-196 70.0/5. 74.5/5.3 IIVDAYGKEEHVLIFD LXAEEVDFXE 253-258 187-196 330-339 3 I .3/5.5 I ACTI A TP? BGL2 EFBI EN02 FBAl GDHl tiPI)/ L 95- IOX GVQyLXX?ITEELi 44.2/6.4 for the Amino acid sequence in one-letter notation a YLPGIXLPDNLVAN 45.1/5.3 74.3/6.1 FEMS Microbiology LYSY MET6 MET1 7 IPPl SAMI’ SSAI/Z Letters 137 (IYY61 I-K expression of these proteins. Proper expression of actin has previously been shown to be of importance during NaCl growth, since act mutants displayed impaired growth in NaCl media [ 181. Actin is slightly regulated at the transcriptional level during adaptation to salt [7]. However, the expression of actin did not display any significant change during exponential growth in saline media. This might indicate cellular independence of actin levels during osmotic stress as long as certain threshold requirements are met. The two proteins Ssal p and Ssa2p (belonging to the HSP 70 family in S. cerevisiae) [19] displayed a slight salt response during adaptation to NaCl [8], while we here observed no difference in expression during exponential growth. The HSP70 homologues Ssblp and Ssb2p have been shown to be involved in the translational process and to be associated with ribosomes [20]. The two isogene products have earlier been identified [lo], and both proteins displayed salt invariant expression. The constancy of the SSB proteins and the translational elongation factor 1p (Efblp) indicate a non-affected protein synthesis during salt growth. Taken together, the constant expression of proteins involved in a number of cellular functions indicate a robust and only slightly altered overall metabolism during growth in salt. SSEI ’ The 2D position of the sequenced proteins are indicated in Fig. 2. ’ Sequences obtained from peptides generated by trypsin digestion and subsequent HPLC fractionation of 2D-PAGE resolved spots, except for the sequences from Fbalp and Eno2p which are from N-terminal sequencing on whole proteins blotted onto PVDF membranes. h The N-terminal methionine in Fbalp and Eno2p was post-translationally removed. ’ Underlined residues are unique to GPDl compared to GPD2. ’ Underlined residues are unique to SAM1 compared to SAM2. ’ Underlined residues are unique to SSEI compared to SSE2. thesis, and the average expression levels of these two proteins were 754 and 222 ppm, respectively. The F, @subunit of the mitochondrial ATPase, Atp2p. also exhibited constant expression (about 290 ppm), indicating a non effected level of mitochondria and/or ATP producing capacity. Assimilation of ammonia and sulfate mediated by Gdhlp and Met1 7p, respectively, also seemed unaffected from the constant 3.3. Levels of glycolytic enzymes during saline growth Studies on cells of S. cerel~isiae grown aerobically in chemostat have indicated rather drastic changes in overall carbon flow under saline conditions [9]. It was reported that (i) the fractional carbon distribution into respiration or fermentation changed, and (ii) up to 16% of the consumed glucose at high dilution rates could not be accounted for by the analysed products. This indicates rather diverse changes in catabolic activities during NaCl stress. Some of the glycolytic enzymes have been shown to be good indicators of metabolic changes, exhibiting metabolism specific expression levels for both transcripts [21] and protein products [22]. Thus, quantitative studies on levels of glycolytic enzymes might reveal overall catabolic changes during saline growth (Fig. 3). One of th e isoenzymes of glyceraldehyde 3-phosphate dehydrogenase (Tdh3p) exhibited the highest level of expression of the glycolytic enzymes J. Norbeck, A. Blomberg / FEMS MicrobiologJl A PDCI ADHl ACT1 0 500 loo0 M&mine 1500 zoo0 25Oil normalized PPM HxK2 FBAl GPDl TDH3 0 loo 300 200 Percent 400 500 Fig. 3. Quantification of 2D-PAGE resolved glycolytic enzymes (indicated in Fig. 3). Actin (ACTI) is included as a point of reference. (A) Quantity expressed in ppm divided by number of methionines in protein, enabling stoichiometric comparison. (B) Quantity expressed as percent, with values in 0 M NaCl medium being set at 100%. Data represent mean of triplicate independent experiments + S.D. in either growth condition and roughly 1% of the incorporated 35S-methionine ended up in this subunit. The incorporation values were normalised to the somewhat different number of methionines in the proteins, and these methionine normalised ppm values revealed Tdh3p to be roughly 30 times more abundant on a mole basis than Actlp (Fig. 3A). During non-saline growth Gpdlp was found in roughly 100 fold lower amounts in relation to Tdh3p, reflecting under these conditions the minor metabolic flux into glycerol compared to into ethanol and biomass. Gpdlp was the only protein of the identified central catabolic enzymes that exhibited a major salt response (Fig. 3B). The enzymes upstream from the triosephosphates, Hxk2p and Fhalp, displayed no significant changes, while downstream especially Tdh3p and Eno2p exhibited decreased expression values. The isoproteins Tdh2p and Enolp displayed no significant change (data not shown). The data on Letters 137 (19961 1-8 I salt induced alterations in protein expression was supported by enzyme activity measurements, where both the glyceraldehyde 3-phosphate dehydrogenase and enolase activities decreased in salt grown cells, the former from 6199 to 5085 mU/ng protein (18% reduction) and the latter from 1048 to 792 (25% reduction). Other determined enzyme activities as those for hexokinase, aldolase, triosphosphate isomerase, and pyruvate decarboxylase exhibited no significant salt dependent variation in activities (data not shown). The rate of glycerol synthesis was earlier indicated to be under the control of the amount of Gpdlp, and a high flux control coefficient was reported for this enzyme [2]. However, the determined flux control coefficient for GPD might be overestimated if additional metabolic changes occur in parallel, especially those alterations in close vicinity to the glycerol pathway [23]. Recent results indicate that at least under certain conditions Gpdlp has low controlling power over the flux to glycerol, since overproduction of the enzyme had no apparent impact on glycerol formation [5]. Apparently, additional enzyme activities have to be regulated besides Gpdlp in order to obtain the massive osmoregulatory production of glycerol. 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