A parallel proteomic and metabolomic analysis of the hydrogen peroxide- and Sty1p-dependent stress response inSchizosaccharomyces pombe

RESEARCH ARTICLE

A parallel proteomic and metabolomic analysis of the

hydrogen peroxide- and Sty1p-dependent stress

response in Schizosaccharomyces pombe

Mark E. Weeks1, John Sinclair1, Amna Butt2, Yuen-Li Chung3, Jessica L. Worthington2,Caroline R. M. Wilkinson2, John Griffiths3, Nic Jones2, Michael D. Waterfield1

and John F. Timms1, 4

1 Ludwig Institute for Cancer Research, University College London, Cruciform Building, London, UK2 Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK3 Department of Basic Medical Sciences, Medical Biomics Centre, St. George’s Hospital Medical School,

Cranmer Terrace, London, UK4 Department of Biochemistry and Molecular Biology, University College London, Cruciform Building, London, UK

Using an integrated approach incorporating proteomics, metabolomics and published mRNAdata, we have investigated the effects of hydrogen peroxide on wild type and a Sty1p-deletionmutant of the fission yeast Schizosaccharomyces pombe. Differential protein expression analysisbased on the modification of proteins with matched fluorescent labelling reagents (2-D-DIGE) isthe foundation of the quantitative proteomics approach. This study identifies 260 differentiallyexpressed protein isoforms from 2-D-DIGE gels using MALDI MS and reveals the complexity ofthe cellular response to oxidative stress and the dependency on the Sty1p stress-activated proteinkinase. We show the relationship between these protein changes and mRNA expression levelsidentified in a parallel whole genome study, and discuss the regulatory mechanisms involved inprotecting cells against hydrogen peroxide and the involvement of Sty1p-dependent stress-acti-vated protein kinase signalling. Metabolomic profiling of 29 intermediates using 1H NMR wasalso conducted alongside the protein analysis using the same sample sets, allowing examinationof how the protein changes might affect the metabolic pathways and biological processesinvolved in the oxidative stress response. This combined analysis identifies a number of inter-linked metabolic pathways that exhibit stress- and Sty1-dependent patterns of regulation.

Received: October 11, 2005Revised: November 17, 2005

Accepted: November 23, 2005

Keywords:

Hydrogen peroxide / Metabolomics / Redox stress

2772 Proteomics 2006, 6, 2772–2796

1 Introduction

The stress responses by which organisms react to environ-mental changes are complex and involve the regulation ofmany genes [1–4]. Studies of stress responses in human celllines has led to apparent contradictory results because oftheir complex regulatory networks, making the use of sim-pler model cell systems more desirable [5]. The fission yeastSchizosaccharomyces pombe is one such model organism forwhich the complete genome has been sequenced [6] andwhich can be easily genetically manipulated [7]. In particular,its stress-activation pathways share homology with higherorganisms and transduce signals to the nucleus, resulting in

Correspondence: Dr. John F. Timms, Ludwig Institute for CancerResearch, University College London, Cruciform Building, GowerStreet, London WC1E 6BT, UKE-mail: [email protected]: 144-20-7679-6334

Abbreviations: CESR, core environmental stress response; Cy2, 3-(4-carboxymethyl) phenylmethyl-3’-ethyloxacarbocyanine halide;Cy3, 1-(5-carboxypentyl)-1’-propylindocarbocyanine halide; Cy5,1-(5-carboxypentyl)-1’-methylindocarbocyanine halide; 2-D-DIGE,2-D difference gel electrophoresis; GPC, glycerophosphocholine;GSH, glutathione; Hsp, heat shock protein; NHS, N-hydroxysuccin-imidyl; ROS, reactive oxygen species; TMAO, trimethylamine-N-oxide; WT, wild-type

DOI 10.1002/pmic.200500741

2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

Proteomics 2006, 6, 2772–2796 Animal Proteomics 2773

altered patterns of gene expression that are critical for theresponse to environmental stresses such as heat, hyper-osmolarity and oxidative stress. A central element of thestress-activated protein kinase cascade in S. pombe is theprotein kinase Sty1p (Spc1/Phh1p), a homologue of mam-malian p38 kinase, which becomes activated in response tosimilar stresses and whose inactivation results in a pleio-tropic sensitivity to stress [8–10]. Sty1p regulates stress-de-pendent gene transcription, at least in part, through thedirect phosphorylation of the b-ZIP transcription factor,Atf1p which anchors Sty1p in the nucleus [11–14]. Impor-tantly, atf1 mutants show defects in Sty1p-dependent tran-scription, but only display a subset of the phenotypes dis-played by sty1 mutants, suggesting that Sty1p controls otheras yet unidentified proteins and pathways [11, 13].

In the study of oxidative stress, it is particularly relevantthat each human cell metabolizes approximately 1012 mole-cules of oxygen per day with 1% of oxygen metabolismresulting in the production of reactive oxygen species (ROS),such as the superoxide anion (O2

-), the hydroxyl radical(?OH) and hydrogen peroxide (H2O2) [15]. Because of theirhigh reactivity, ROS can bring about cellular damage to var-ious macromolecules. For example, oxidative DNA damagecan alter purine and pyrimidine bases, as well as cleave thephosphodiester DNA backbone leading to genetic mutation[15], whilst oxidation of protein cysteine thiol groups cancause intermolecular protein cross-linking and enzymeinactivation which potentially lead to cell death. Importantly,ROS have been implicated in a number of human diseases[16–18], and for example, it is recognized that many types ofcancer cells exhibit increased production of H2O2 that hasbeen linked to proliferative signalling and tumourigenesis[19–22], although low levels of H2O2 appear to be required fornormal proliferative signalling [23].

Cells have evolved a number of mechanisms to counteractoxidative damage including the direct reversal of mutationsthrough mismatch repair and DNA excision pathways [24], theswift elimination of ROS and the reversal of oxidative damage.The latter are achieved through the induction of antioxidantand redox enzymes such as catalase, superoxide dismutase,peroxidases and thioredoxin, through the maintenance of highlevels of molecular scavengers such as glutathione (GSH) andascorbic acid and by S-thiolation, whereby oxidized thiolgroups form mixed disulphides with GSH which are thenregenerated by glutaredoxins and GSH reductase [25, 26]. Thiolgroups are reported to have numerous roles within the cell andtheir redox states affect the activity and structure of many pro-teins including transcription factors, proteases and phospha-tases. It is not surprising, therefore, that all organisms containregulatory machinery whose purpose is to maintain the redoxstatus of SH groups in both proteins and low-molecular-massthiols [27–29]. In yeast, it has been shown that basal protein S-thiolation is maintained at a low level, but is increased duringoxidative stress where it serves an adaptive function by repro-gramming metabolism and protecting protein synthesisagainst irreversible oxidation [30, 31].

Traditionally, molecular and cellular studies have tendedto concentrate on individual genes and their products, al-though more recently, several microarray studies havedetailed the global genomic responses of yeast to a variety ofstresses including oxidative stress [1–3, 32]. In addition, theglobal analysis of gene products by proteomics has alloweda systematic overview of thousands of proteins at the sametime. 2-DE is one of the most widely used proteomicseparation methods and is often employed for the analysisof differential protein expression across biological samples[33, 34]. A significant improvement came with the intro-duction of 2-D-difference gel electrophoresis (2-D-DIGE), inwhich several samples can be codetected on the same 2-DEgel after differential covalent labelling with matched fluo-rescent tags [35–37]. By alleviating the problems of gel-to-gelvariation, the 2-D-DIGE strategy provides improved accu-racy in quantifying protein differences between samples. Afurther level of global investigation has been realized withthe advent of metabolomics where, for example, NMRspectroscopy can be used to measure changes in the levelsof multiple low-molecular weight metabolites across bio-logical samples. Moreover, by integrating data from metab-olite analysis, gene expression profiling and proteomics, keyperturbed metabolic pathways can be identified providing agreater understanding of the biological processes involvedin the response to environmental changes. Metabolomicshas already been used to study toxicological mechanismsand disease processes and offers great potential as a meansof investigating the complex relationship between stressand metabolism [38, 39].

In this study, we have used 2-D-DIGE to examine prote-omic changes generated by the application of peroxide stressto fission yeast and have compared this data with thatobtained from transcriptional and metabolomic profilingacquired under identical growth conditions. The degree towhich each data set correlates and how the changes relate toaltered biological function is shown and discussed. Further-more, we have examined the effects of the loss of the stress-dependent MAP kinase Sty1 on the peroxide stress responseand have been able to differentiate altered gene expressionstates and metabolic pathways between the unstressed andperoxide-stressed cell types.

2 Materials and methods

2.1 Growth and hydrogen peroxide treatment of

S. pombe

Yeast strains used were wild-type (WT) strain 972 h2 and anisogenic Sty1p-deleted mutant strain sty1D (sty1::ura41 ura4-D18 h2). Two litre cultures of WT or sty1D in yeast extract(YE) medium [40] were grown to mid-exponential phase(56106 cells/mL), and subjected to oxidative stress by addi-tion of 0.5 mM H2O2 for 60 min or left untreated. Cells wereharvested by centrifugation (580g), washed twice in water


2774 M. E. Weeks et al. Proteomics 2006, 6, 2772–2796

and re-suspended in 1 mL of grinding/resuspension buffer(10 mM HEPES–HCl pH 7.4, 0.1% IGEPAL CA-630 (pre-viously NP 40), 2 mM EDTA, 4 mg/mL leupeptin, 4 mg/mLaprotinin and 5 mg/mL pepstatin A). Cell suspensions wereimmediately frozen by addition of liquid N2.

2.2 Cell lysis and CyDye-labelling of protein extracts

Frozen cells were broken by grinding for 20 min underliquid N2 in a RM100 mortar grinder (Retsch, Germany) asdescribed [41] and ground lysates stored at 2807C. ForNHS-Cy dye labelling 100 mL aliquots of the lysates weresuspended in 2-D buffer (8 M urea, 2 M thiourea, 4%CHAPS, 0.5% IGEPAL CA-630, 10 mM Tris-HCl pH 8.3)without reductant to give a sample volume of 600 mL. Sam-ples were homogenized by passing through a 25-gaugeneedle six times prior to agitation at room temperature for10–15 min. Insoluble material was removed by centrifuga-tion (12 0006g/10 min/47C). Protein concentrations weredetermined using Coomassie Protein Assay Reagent(Pierce) and a BSA standard curve with four replicate assaysperformed per sample.

A sample amount of 150 mg was aliquoted into tubes forNHS-Cy dye labelling. Equal amounts of protein from eachsample were also mixed to create an internal standard to belabelled with Cy2 and run on each gel. NHS-Cy2 was pur-chased from GE Healthcare (Amersham, UK) whilst NHS-Cy3 and Cy5 were synthesized in-house and stored asdescribed [42]. They are structurally identical to the com-mercially available compounds and highly pure. Sampleswere labelled by addition of 4 pmol of NHS-Cy dye permicrogram of protein (600 pmol/150 mg) and incubation onice in the dark for 30 min. Reactions were quenched with a20-fold molar excess of L-lysine to dye and further incubationon ice in the dark for 10 min. Cy3 and Cy5 were randomlyassigned to biological triplicate samples from separatelygrown and treated cultures for the four conditions underinvestigation. Cy3- and Cy5-labelled samples were mixedappropriately (Table 1) and the same amount (150 mg) of theCy2-labelled pool was added. Samples were reduced withDTT at a final concentration at 65 mM. Carrier ampholines/pharmalyte mixture was added to a final 2% v/v and bromo-phenol blue added. The volume was adjusted to 450 mL withlysis buffer and the samples were agitated prior to cen-trifugation at 12 0006g for 10 min.

Table 1. Typical experimental design showing allocation of Cydye labelling of biological replicates across gels. Cy2was used to label an equal pool (by protein amount) ofall sample replicates

1 2 3 4 5 6

Cy2 Pool Pool Pool Pool Pool PoolCy3 sty1D-60’ WT-60’ WT-0’ sty1D-60’ WT-0’ WT-60’Cy5 WT-0’ Sty1D-0’ WT-60’ Sty1D-0’ Sty1D-0’ Sty1D-60’

2.3 2-DE, fluorescence imaging and image analysis

2-DE was carried out essentially as previously described [43]using 24 cm pH 4–7 L and pH 3–10 NL IPG strips (GEHealthcare) and 12% homogenous SDS-PAGE gels bonded tolow-fluorescence glass plates. Gels were run in Ettan12 geltanks (GE Healthcare) at 2.2 mA per gel and 167C until the dyefront had run off the bottom. All steps were carried out in adedicated clean room. Gels were scanned between glass platesusing a Typhoon 9400 variable mode imager and Image-Quant software (both GE Healthcare). The photomultipliertube voltage was adjusted on each channel (Cy2, Cy3 and Cy5)for preliminary low-resolution scans to give maximum pixelvalues within 5–10% for each Cy-image, but below the satura-tion level. These settings were then used for high-resolution(100 mm) scans of all gels. Images were exported as tiff files forimage analysis. Images were then curated and analysed usingDeCyder software v5.0 (GE Healthcare) essentially as pre-viously described [43]. Here comparison of test spot volumes(Cy3 or Cy5 labelled) with the corresponding standard spotvolume (Cy2 labelled) gave a standardized abundance for eachmatched spot and values were averaged across biological tri-plicates. Only spots displaying a �1.5 average-fold increase ordecrease in abundance between each condition, matchingacross all gel images and having p values ,0.05 were selectedfor identification.

2.4 Spot picking, tryptic digestion and protein

identification

All gels were poststained with colloidal Coomassie Blue G-250 (CCB) and imaged as previously described [43]. CCB-stained images were then matched with the correspondingfluorescent images using DeCyder and a pick list of coordi-nates relative to two reference markers (stuck to plates atcasting) was generated for robotic excision from gels usingan Ettan automated spot picker (GE Healthcare). Spots werecollected in 96-well plates, proteins digested with trypsin andresultant peptides extracted as described previously [43].Extracts were dried, resuspended in 5 mL of 20 mM ammo-nium phosphate and spotted (0.5 mL/sample) onto MALDItarget plates with 1 mL of saturated 2,5-dihydroxybenzoic acid(DHB) in 20 mM ammonium phosphate using the drieddroplet method.

MALDI-TOF MS with peptide mass fingerprinting wasused for protein identification. MALDI mass spectra wereacquired using an Ultraflex mass spectrometer (Bruker Dal-tonics) in the reflector mode externally calibrated using an‘in-house’ mixture of standard peptides. Spectra were ana-lysed in FlexAnalysis (Bruker Daltonics). Where possiblethe spectra were internally calibrated using the trypsin auto-lysis peaks at m/z 842.51 and m/z 2211.10. The SNAP algo-rithm in FlexAnalysis was used to pick up to the 100 mostprominent peaks in the mass range m/z 600–5000. Masslists were extracted from the Ultraflex data files using an in-house Perl script, UltraMassList (http://www.ludwig.ucl.



ac.uk/bachem_html/software.htm). Another Perl script Com-monContam (http://www.ludwig.ucl.ac.uk/bachem_html/software.htm), based on the software of Schimdt et al. [44], wasused to identify masses common to all peak lists (i.e. matrixpeaks, trypsin peaks) above a user-defined threshold. Com-mon masses were removed and the resulting peptide massfingerprints were searched against an up-to-date NCBI andS. pombe database using MASCOT with the automated MAS-COT Daemon (Matrix Sciences v1.9.0 or later v.2.0). A positiveidentification was accepted when a minimum of six peptidemasses matched to a particular protein (mass error of650 ppm allowing one missed cleavage), matched peptidesrepresented�25% of the protein sequence, the MOWSE scorewas over the threshold score (p = 0.05), the hit appeared in thetop five hits in both database searches and the gel-based mo-lecular weight was in general agreement with the predictedmolecular weight of the identified protein.

2.5 Deletion of SPACUNK4.17 and SPAC23H3.15c

S. pombe gene deletions were constructed using establishedmethods [45]. All oligonucleotide sequences used are avail-able upon request. To test for sensitivity to H2O2, 8 mL ofserial five-fold dilutions of cells were plated on YE mediawith and without H2O2 and grown for 4 days at 307C prior toexamination of growth. The starting concentration of cellswas 46106 cells/mL. The sty1D and atf1D mutants, whichhave known sensitivity to H2O2, were used as controls.

2.6 1H NMR analysis of cellular metabolites

Ground yeast lysate (corresponding to 7.5 mg of total protein)from five biological replicates were extracted in 6% perchloricacid as previously described [46]. Neutralized extracts werefreeze-dried and reconstituted in 1 mL of D2O, and the extracts(0.5 mL) were placed in 5 mm NMR tubes. 1H NMR of cellextracts was performed on a Bruker 600 MHz NMR system(pulse angle: 907; repetition time: 5 s). The water resonancewas suppressed by gated irradiation centred on the waterfrequency. Sodium 3-trimethylsilyl-2,2,3,3-tetradeuteropro-pionate (25 mL, 10 mM) was added to the samples for chemicalshift calibration and quantification. All cell extract spectrawere acquired under identical conditions.

3 Results and discussion

3.1 2-D-DIGE and MS analysis of H2O2 and

Sty1p-dependent protein expression

Lysates of control and H2O2-treated WT and sty1D mutantS. pombe cells were compared as biological triplicates using2-D-DIGE allowing the averaging of possible variations ingrowth conditions, sample processing and gel running. Atreatment of 0.5 mM H2O2 for 60 min (60’) was chosen as anoxidative stress since this is known to induce stress genes

while causing minimal cell death and was the concentrationused in a previous parallel global transcriptional analysis [1].It should be noted that the transcriptional responses ofS. pombe vary with H2O2 concentration [47], and here wedescribe the responses to an intermediate level of oxidant.Samples were labelled with either Cy3 or Cy5 and run againsta Cy2-labelled pool of all samples, run on all gels as an inter-nal standard for improved cross-gel matching and quantita-tion [35, 48]. Three experiments were conducted using pH 3–10 nonlinear pH range IPG strips and two with pH 4–7separations. For each experiment DeCyder software was usedto find protein features displaying a .1.5 average fold-change in abundance in WT and sty1D cells in response toH2O2 or between the WT and sty1D cells. Figure 1 shows atypical master gel as an overlay of images (WT-60’ vs. sty1D-60’) with the position of all proteins for which unambiguousidentifications were obtained by MALDI-TOF MS and pep-tide mass fingerprinting.

A total of 260 protein isoforms were identified whichdisplayed significant differences in expression in one ormore of the comparisons (WT-60’ vs. WT-0’, sty1D-60’ vs.sty1D-0’, sty1D-0’ vs. WT-0’ and sty1D-60’ vs. WT-60’). Thecomplete data set is shown in Table S1 of SupplementaryMaterial. Average abundance ratios (based on fluorescenceintensity) for 836 data points out of a possible 1040 wereobtained. Unrecorded data points fell outside the selectionparameters (i.e. not matching across all gels or had p values.0.2). There were 777 data points with significant p values of,0.05. The 260 proteins represented 158 different geneproducts demonstrating a high degree of PTMs or proteo-lysis. Within the entire sample set, 33 spots yielded dataindicating that they contained two proteins with one spotyielding three. In these instances, the quantitative data can-not be assigned to an individual gene product, although thetarget of altered expression could be inferred in some casesfrom correlative changes in mRNA levels (see below). Acomparison of predicted vs. gel-based Mr and pI was con-ducted (Fig. 2). The correlation of Mr was reasonable and wasused as an additional level of confidence for protein identifi-cations, although it was skewed for higher Mr gene productsas these are under-represented on 2-D gels. The pIs corre-lated much less well, again demonstrating a high degree ofPTMs that affect pI (see below).

Figure 3A shows the number and direction of regulationof proteins changing due to H2O2 treatment for each strainand how these overlap, whilst Fig. 3B shows the number anddirection of regulation between cell types in the absence andpresence of stress. The greatest number of differences wasobserved when comparing WT-60’ with sty1D-60’ indicating acritical role of Sty1p in the stress response. Indeed, 47 pro-tein isoforms were up-regulated in WT in response to H2O2

compared to 14 for sty1D, whilst there were a similar numberof down-regulated proteins. Differences were also observedbetween unstressed WT and sty1D with 60 proteins (51 geneproducts) requiring Sty1p for basal expression. A number ofgene products were also de-repressed in sty1D showing that



Figure 1. Master gel image showing the positions of S. pombe protein isoforms displaying peroxide and/or Sty1p-dependent changes inexpression. Figure shows an overlay of two pseudo-coloured images of a WT-60’ sample repeat (Cy3, red) and a sty1D-60’ sample repeat(Cy5, blue) created in Photoshop v7. Images were taken from a bonded pH 3–10 NL IPG, 24 cm624 cm61 mm, 12.5% bis/acrylamide gel.Numbers and arrows indicate positions of differentially expressed spots selected by DeCyder software that were identified with high con-fidence by MALDI-TOF MS (see Table S1).

Figure 2. (A) Graphical compar-ison of predicted vs. experimen-tally/gel-determined Mr and (B)pIs for the identified protein iso-forms. Predicted pI and Mr weretaken from databases. Gel-based pI and Mr were calculatedusing DeCyder software basedon selected reference proteinswhere gel-based values andpredicted values were in agree-ment.



Figure 3. Schematic representationof regulated isoform expression. (A)Venn diagram showing the numbersof H2O2-dependent up- and down-regulated isoforms (and gene prod-ucts) in WT and sty1D cells and theiroverlapping patterns of expression.(B) Venn diagram showing the num-bers of Sty1p-dependent up- anddown-regulated isoforms (and geneproducts) in untreated and H2O2-treated cells. Isoforms were takenfrom Table S1 and were included ifthey displayed a .1.5 average-foldchange in abundance (n = 3;p , 0.05).

Sty1p can also repress gene expression in agreement withthe parallel whole-genome transcriptional analysis [1]. How-ever, it is important to note that Sty1p may also alter the iso-form distribution of gene products through induced PTMs.Notably, 48% of all differentially expressed proteins wereregulated by H2O2 in a similar way in the WT and sty1Dstrains, showing that despite the central role of Sty1p instress-activated signalling, many cellular responses do notrequire its function.

The identified proteins were also grouped using k-meansand hierarchical clustering enabling us to rapidly view co-regulated isoforms displaying H2O2- and/or Sty1p-dependencyand to assess whether groups of functionally related proteinswould cocluster (Figs. 4 and S1). Proteins showing potent,Sty1p-dependent H2O2 induction (Fig. 4A) included catalase,GSH S-transferase II, thioredoxin reductase, malate dehy-drogenase, phosphoglycerate dehydrogenase, elongation fac-tor 2, protein tyrosine phosphatase/Pyp3p and three geneproducts of unknown function (an aldo/keto reductase(SPBC215.11c), brefeldin A-resistance protein p20(SPAC3C7.14c) and a hypothetical serine-rich protein(SPAC23H3.15c)). In contrast, Fig. 4B shows a cluster of 20proteins that were de-repressed in the sty1D mutant but rela-tively unaffected by stress. Prominent in this group were mem-

bers of the heat shock protein (Hsp) family (Hsp90 homologueSwo1, Hsp70 family Ssa2, heat shock cognate protein Hsc1,Hsp homologue Pss1 and Hsp70 family Ssc1). This suggeststhat loss of Sty1p may put cells in a permanent state of stress.Another large de-repression (17-fold) was seen for the leucinebiosynthetic enzyme 3-isopropylmalate dehydrogenase, whichwas also down-regulated by H2O2 treatment.

3.2 Functional classification of identified proteins

Each identified protein was assigned a functional classifica-tion based on the gene ontology annotation in the S. pombedatabase (http://www.genedb.org/genedb/pombe/index.jsp)and pathway assignment detailed on the S. pombe KyotoEncyclopaedia of Genes and Genomes database (KEGG,http://www.genome.jp/kegg/kegg2.html). The functionalityof all proteins in our data set is represented in Fig. 5. Themajority (57%) of differentially regulated protein isoformsfell within four functional groups (glycolysis, amino acidmetabolism, molecular chaperones and protein synthesis),while 11% were of unknown function. The remaining 32%were represented in 15 different functional categories. Pro-teins involved in glycolysis (see Table 2) represented thelargest functional group with 50 isoform entries (14 gene



Figure 4. k-Means and hierarchicalclustering of protein differences.Upper panel shows a cluster of pro-tein isoforms that were induced byH2O2 in a Sty1-dependent manner.Lower panel shows isoforms whoseexpression was induced by loss ofSty1p (i.e. de-repressed). Clusteringof all identified isoforms was carriedout on the log-transformed 2-D-DIGE ratios (Table S1) using adownloadable version of Multi-Experiment Viewer (MeV) (http://www.tm4.org/mev.html) from TheInstitute for Genome Research(TIGR).

products), indicating a sensitivity of this central metabolicpathway to H2O2 and/or loss of Sty1p. In general in WTcells,the glycolytic enzymes in the upper half of the pathway wereup-regulated while those in the lower half were down-regu-lated. This suggests that glycolytic flux is redirected, perhapsto the pentose phosphate pathway as evidenced by changesin the expression of several pentose phosphate pathwayenzymes (phosphoglucomutase, ribose 5-phosphate isomer-ase, transketolase, transaldolase, 6-phosphogluconate dehy-drogenase and glucose-6-phosphate 1-dehydrogenase/Zwf1p), some of which were induced in a Sty1p-dependent

manner. Previous studies of oxidative stress in S. cerevisiaealso came to this conclusion [30, 49], although the study byShenton et al. showed that H2O2 inhibited the activities ofglyceraldehyde-3-phosphate dehydrogenase, enolase andalcohol dehydrogenase through reversible S-thiolation ratherthan through altered expression and there was little effect onglucose-6-phosphate dehydrogenase/Zwf1 or 6-phosphoglu-conate dehydrogenase activity, enzymes that catalyseNADPH production via the pentose phosphate pathway.Thus, inhibition of glycolytic flux alone was proposed toresult in glucose equivalents entering the pentose phosphate



Figure 5. Functional classification and distribution of all identified protein isoforms. Gene Ontology (GO) annotation terms for biologicalprocess and molecular function were taken from the S. pombe gene database (http://www.genedb.org/genedb/pombe/index.jsp), theKyoto Encyclopaedia of Genes and Genomes (KEGG) Pathways Database (http://www.genome.jp/kegg/kegg2.html) and the Swiss-Prot/TrEMBL protein database (http://us.expasy.org/sprot/). Unknown proteins include those which have no ascribed function, but may fall intoa putative enzyme class based on sequence homology.

pathway for the generation of NADPH and reductive capacity[30]. Phosphoglucomutase (induced in a Sty1-dependentmanner) is also required for the synthesis of the knownstress-protectant sugar trehalose, in agreement with obser-vations made in S. cerevisiae [49]; however, other enzymes inthis pathway were not found.

Amino acid metabolism also featured heavily with 42identified isoforms (25 gene products), as did protein syn-thesis (27 identifications) and protein degradation/proces-sing (9 identifications). These results suggest that globalalterations in protein turnover may occur in response toH2O2 through the regulation of amino acid synthesis, pro-tein synthesis and degradation. Although oxidative stress hasbeen shown to inhibit protein synthesis in S. cerevisiae [30],here, no broad pattern of coregulation could be discernedwhich might suggest a global switch in translational activity.For example, whilst isoforms of five eukaryotic translationinitiation factors were identified (eIF2a, eIF2Bg, eIF3 RNA-binding subunit, eIF3 p39 subunit and eIF4A), they dis-played different patterns of expression in response to H2O2,as did the seven ribosomal proteins identified. Molecularchaperones appeared 29 times, the majority of which wereHsps (Table 3). These changes may be required to aid proteinfolding in a more oxidizing environment or for the removalof aggregated or misfolded proteins; however, there was nocommon pattern of regulation apparent.

Of particular interest to this study were proteins knownto be involved in redox regulation and it is not surprising thatthere were 32 oxidoreductases in the 158 gene productsidentified. The redox enzymes catalase, thioredoxin reduc-tase, GST2 and thioredoxin peroxidase were all up-regulatedin WT cells exposed to H2O2, but were not induced in sty1D

cells (Table 3). This demonstrates the critical role played bySty1p signalling in the induction of antioxidants for removalof cellular H2O2 and protection from oxidative stress. Inter-estingly, one of these antioxidant enzymes, the highly con-served 2 cys-peroxiredoxin thioredoxin peroxidase (Tpx1p),was recently shown to directly activate Sty1p by a mechanisminvolving the formation of a peroxide-induced disulphidecomplex between Tpx1p and Sty1p, with overexpression ofTpx1p resulting in hyperactivation of Sty1p [50]. Thus, thereappears to be a feedback mechanism whereby Sty1 activity isrequired for the induction of one of its own activators. Nota-bly, GSH synthetase large chain/Gsa1p (SPAC3F10.04) wasinduced by H2O2 in both cell types (Table 3) and may berequired to increase GSH production, whilst Sty1p-depend-ent glucose-6-phosphate dehydrogenase induction andinduction of succinate semialdehyde dehydrogenase wouldalso increase NADPH reducing equivalents for reduction ofGSH and protein. Several gene products of unknown func-tion also displayed Sty1p-dependent induction (aldo/ketoreductase SPBC215.11c, brefeldin A-resistance protein p20SPAC3C7.14c, sugar oxidoreductase SPACUNK4.17 and hy-pothetical proteins SPAC23H3.15c, SPCC777.06c,SPAC1002.18), suggesting that they too may be antioxidants.To further investigate the function of two of these unchar-acterized gene products, whose mRNAs were also induced(namely hypothetical serine-rich protein SPAC23H3.15c andsugar oxidoreductase SPACUNK4.17), we constructedmutant strains lacking these genes and tested their sensitiv-ity to H2O2. Figure 6 shows that growth of the sty1D mutant,and to a lesser extent an atf1D mutant, was as expected, sen-sitive to H2O2. However, there was no apparent effect ofdeleting SPAC23H3.15c or SPACUNK4.17. Thus, being



Table 2. Differentially regulated enzymes involved in glycolysis. Proteins are organized alphabetically with spot numbers from DeCyderanalysis. Spots containing two gene products are labelled a and b

Spotno.

Proteinname

ORFname

E.C.no.

Average fold-change pI(gel)

Mr

(gel)pI(pred)

Mr

(pred)WT-60’vs. WT-0’

sty1D-60’vs. sty1D-0’

sty1D-0’vs. WT-0’

Sty1D-60’vs. WT-60’

794a Alcohol dehydrogenase 1, adh1a) SPCC13B11.01 1.1.1.1 21.61 21.28 21.21 – 7.37 51 910 6.46 37 9401012 Alcohol dehydrogenase 1, adh1 a) SPCC13B11.01 1.1.1.1 1.32 3.11 21.31 1.81 7.14 43 840 6.46 37 940

550 Alcohol dehydrogenase 1, adh1a) SPCC13B11.01 1.1.1.1 1.54 1.58 2.01 2.06 4.58 61 820 6.46 37 9401068a Alcohol dehydrogenase 1, adh1a) SPCC13B11.01 1.1.1.1 2.3 – 1.71 21.17 7.15 41 870 6.46 37 940

839 Alcohol dehydrogenase 4, adh4 SPAC5H10.06c 1.1.1.1 21.15 21.17 21.5 21.52 7.42 50 170 6.46 45 5001057 Alcohol dehydrogenase 4, adh4 SPAC5H10.06c 1.1.1.1 21.14 21.23 21.48 21.59 7.4 43 000 6.46 45 500

710a Alcohol dehydrogenase 4, adh4 SPAC5H10.06c 1.1.1.1 1.15 – 21.74 22.01 7.36 55 980 6.46 45 500939a Alcohol dehydrogenase 4, adh4 SPAC5H10.06c 1.1.1.1 – – – 21.69 7.42 46 100 6.46 45 500770 Dihydrolipoamide acetyltransferase,

E2 of pyruvate dehydrogenasecomplex

SPCC794.07 2.3.1.12 1.79 21.13 1.28 21.58 5.4 60 150 5.6 56 300

717 Enolasea) SPBC1815.01 4.2.1.11 21.17 – 21.61 – 7.22 54 910 6.23 47 580710b Enolasea) SPBC1815.01 4.2.1.11 1.15 – 21.74 22.01 7.36 55 980 6.23 47 580681 Enolasea) SPBC1815.01 4.2.1.11 1.18 2.45 21.31 1.59 6.52 56 230 6.23 47 580698 Enolasea) SPBC1815.01 4.2.1.11 1.29 2.71 – 2.15 7 55 980 6.23 47 580853b Enolasea) SPBC1815.01 4.2.1.11 1.8 1.35 3.34 2.51 6.68 49 800 6.23 47 580691 Enolasea) SPBC1815.01 4.2.1.11 – 1.75 21.41 – 6.85 55 900 6.23 47 580658 Enolasea) SPBC1815.01 4.2.1.11 – – 21.46 21.87 7.24 56 980 6.23 47 580

1035 Fructose 1,6-bisphosphate aldolasea) SPBC19C2.07 4.1.2.13 1.73 2.16 21.47 21.18 6.45 42 940 5.92 39 7201275 Fructose 1,6-bisphosphate aldolasea)

(fragment?)SPBC19C2.07 4.1.2.13 21.51 21.11 3.31 4.49 6.4 30 000 5.92 39 720

980 Glyceraldehyde 3-phosphatedehydrogenase 1a)

SPBC32F12.11 1.2.1.12 21.34 – 2.9 3.91 4.76 45 220 6.24 36 020


SPBC32F12.11 1.2.1.12 21.21 21.26 1.6 1.54 4.88 35 730 6.24 36 020

1125a Glyceraldehyde 3-phosphatedehydrogenase 1a)

SPBC32F12.11 1.2.1.12 1.2 21.35 21.43 22.31 7.22 39 580 6.24 36 020


SPBC32F12.11 1.2.1.12 1.28 2.26 – 169 6.93 39 870 6.24 36 020


SPBC32F12.11 1.2.1.12 1.3 2.52 1.1 2.13 6.41 39 810 6.24 36 020


SPBC32F12.11 1.2.1.12 1.44 21.92 – 23.54 7.48 39 810 6.24 36 020

1135b Glyceraldehyde 3-phosphatedehydrogenase 1a)

SPBC32F12.11 1.2.1.12 1.56 1.45 2.09 1.94 6.58 39 350 6.24 36 020


SPBC32F12.11 1.2.1.12 1.58 21.57 21.45 23.58 7.42 39 760 6.24 36 020


SPBC32F12.11 1.2.1.12 – 21.06 5.19 5.75 6.79 39 350 6.24 36 020


SPBC32F12.11 1.2.1.12 – 1.69 1.78 2.19 6.93 38 540 6.24 36 020


SPBC32F12.11 1.2.1.12 – 22.07 21.35 22.41 7.31 39 870 6.24 36 020


SPBC354.12 1.2.1.12 1.2 21.35 21.43 22.31 7.22 39 580 7.7 35 823


SPBC354.12 1.2.1.12 1.56 1.45 2.09 1.94 6.58 39 350 7.7 35 823


SPBC354.12 1.2.1.12 1.58 21.57 21.45 23.58 7.42 39 760 7.7 35 823


SPBC354.12 1.2.1.12 – 21.06 5.19 5.75 6.79 39 350 7.7 35 823


SPBC354.12 1.2.1.12 – 1.69 1.78 2.19 6.93 38 540 7.7 35 823



Table 2. Continued

Spotno.

Proteinname

ORFname

E.C.no.

Average fold-change pI(gel)

Mr

(gel)pI(pred)

Mr

(pred)WT-60’vs. WT-0’

sty1D-60’vs. sty1D-0’

sty1D-0’vs. WT-0’

Sty1D-60’vs. WT-60’

518 Hexokinase 1a) SPAC24H6.0 2.7.1.1 1.55 1.22 21.21 21.53 4 62 740 5.02 54 250124 Pfk1, 6-phosphofructokinase

(predicted)SPBC16H5.02 2.7.1.11 21.16 21.15 1.51 1.52 6.03 86 150 5.8 103 460

118 Pfk1, 6-phosphofructokinase(predicted)

SPBC16H5.02 2.7.1.11 – – 1.32 1.54 5.93 86 410 5.8 103 460

342b Phosphoglucomutase precursor(predicted)

SPBC32F12.10 5.4.2.2 1.3 21.34 21.33 22.32 6.09 60 900 6.09 60 900

833 Phosphoglycerate kinase SPBC14F5.04c 2.7.2.3 1.16 – 21.95 22.4 8.01 50 090 8.33 44 2801224a Phosphoglycerate kinase SPBC14F5.04c 2.7.2.3 1.57 1.17 21.21 21.62 7.33 35 890 8.33 44 280

829 Phosphoglycerate kinase SPBC14F5.04c 2.7.2.3 – – 21.68 22.55 8.15 50 390 8.33 44 280850 Phosphoglycerate kinase SPBC14F5.04c 2.7.2.3 – 21.21 22.19 22.81 8.5 49 580 8.33 44 280867 Phosphoglycerate kinase SPBC14F5.04c 2.7.2.3 – – 21.94 22.17 8.65 49 650 8.33 44 280830 Phosphoglycerate kinase SPBC14F5.04c 2.7.2.3 – – – 22.31 8.76 50 240 8.33 44 280

1479 Phosphoglycerate mutasea) SPAC26F1.06 5.4.2.1 21.04 21.58 21.31 21.99 6.9 23 810 6.92 23 810794b Pyruvate dehydrogenase E1 alpha

subunitSPAC26F1.03 1.2.4.1 21.61 21.28 21.21 – 7.37 51 910 8.34 45 450

795 Pyruvate dehydrogenase E1 alphasubunit

SPAC26F1.03 1.2.4.1 2.04 3.29 21.71 – 6.93 51 910 8.34 45 450

800 Pyruvate dehydrogenase E1 alphasubunit

SPAC26F1.03 1.2.4.1 – – 21.59 21.74 7.13 51 830 8.34 45 450

1231 Pyruvate kinase (fragment?) SPAC4H3.10c 2.7.1.40 3.04 – 2.64 – 7.02 35 310 8.18 56 050708a Pyruvate kinase (predicted) SPAC4H3.10c 2.7.1.40 – 21.13 2.06 1.83 4.89 55 070 8.18 56 050

a) Identifications found in multiple experiments. ORF names and Enzyme Commission (EC) number were taken from the S. pombe genedatabase (http://www.genedb.org/genedb/pombe/index.jsp) and from the Kyoto Encyclopaedia of Genes and Genomes (KEGG) Path-ways Database (http://www.genome.jp/kegg/kegg2.html). Average-fold differences in expression between H2O2-treated and untreatedWT and sty1D cells and between WT and sty1D cells were taken from DeCyder analyses of triplicate samples run in four independent 2-D-DIGE experiments. Only changes are reported where spots matched across all gels within an experiment and fold-changes had pvalues ,0.05 (except those in italics where p values were .0.05 and ,0.2). Isoforms up- and down-regulated by .1.5-fold are high-lighted in dark grey and light grey, respectively. Experimental pI and Mr (gel) were calculated in DeCyder using pI and Mr of knownproteins as references. Multiple isoforms of proteins are listed in order of increasing pI. Predicted pI and Mr were taken from databases.Proteins were denoted with (fragment?) where gel Mr was significantly lower than predicted.

highly induced by H2O2 does not necessarily indicate that agene is essential for a protective response to oxidative stress,and some redundancy must therefore exist.

3.3 Regulated expression, isoform distribution and

post-translational modifications

As well as changes in gene expression, the observed regu-lated protein expression in response to H2O2 or loss of Sty1pis likely to involve that post-transcriptional regulatory mech-anisms such as altered translation, degradation and PTMs.Indeed, this was evidenced by the existence of multiple iso-forms of the same gene products; of 158 gene productsidentified, 44 occurred as multiple isoforms. For example,glyceraldehyde-3-phosphate dehydrogenase was identified as16 different isoforms, consisting of a mixture of the tdh1 andtdh2 gene products (Table 2). Upon H2O2 stress in the sty1Dmutant, several basic isoforms were down-regulated, whilst

the acidic forms were up-regulated indicative of induciblePTMs that affect pI. Indeed, some isoforms (including thoseof enolase and fructose bisphosphate aldolase) had matchedpeptide pairs separated by 79 Da (results not shown) indica-tive of phosphorylation. However, pI shifts may also be dueto the irreversible oxidation of cysteinyl thiols to the R-SO2

2

and R-SO32 forms as reported for peroxiredoxins in redox

stressed cells [51, 52]. This oxidative damage is likely to bemore prevalent in stressed sty1D cells, where redox protectiveenzymes such as catalase and thioredoxin peroxidase are notinduced (Table 3).

Several proteins were also identified as putative frag-ments by virtue of the sequence coverage obtained by MSand their unexpected gel positions. For example, poly(A)-binding protein/Pab1p involved in RNA processing (poly(A)shortening and translation initiation) was identified as threeisoforms; two as the likely whole proteins which were down-regulated in sty1D and one as a lower Mr fragment which was


2782 M. E. Weeks et al. Proteomics 2006, 6, 2772–2796Tab

le3.

Dif

fere

nti

ally

reg

ula

ted

pro

tein

iso

form

so

fin

tere

st.E

xpre

ssio

nd

ata

are

sho

wn

for1

13se

lect

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ofo

rms,

som

eo

fwh

ich

are

dis

cuss

edin

the

text

.An

no

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on

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he

tab

leis

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rTa

ble

2,ex

cep

tth

atsh

aded

pro

tein

nam

esin

dic

ate

tho

seid

enti

fied

inp

H4–

7g

els

and

on

tolo

gy

ann

ota

tio

nte

rms

for

bio

log

ical

pro

cess

and

mo

lecu

lar

fun

ctio

nw

ere

take

nfr

om

the

S.

po

mb

eg

ene

dat

abas

e(h

ttp

://w

ww

.gen

edb

.org

/gen

edb

/po

mb

e/in

dex

.jsp

),th

eS

wis

s-P

rot/

TrE

MB

Lp

rote

ind

atab

ase

(htt

p://

us.

exp

asy.

org

/sp

rot/

)an

dfr

om

the

Kyo

toE

ncy

clo

pae

dia

of

Gen

esan

dG

eno

mes

(KE

GG

)P

ath

way

sD

atab

ase

(htt

p://

ww

w.g

eno

me.

jp/k

egg

/keg

g2.

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l).S

had

edb

iolo

gic

alp

roce

ss/m

ole

cula

rfu

nct

ion

ind

icat

esp

rote

ins

wit

hkn

ow

nro

les

inre

do

xre

gu

lati

on

or

po

sses

sin

gkn

ow

no

rp

red

icte

do

xid

ore

du

ctas

eac

tivi

ty

Spot

no.

Prot

ein

nam

eOR

Fna

me

E.C.

no.

Aver

age

fold

-cha

nge

pI,

gel

Mr

(gel

)pI (p

red)

Mr

(pre

d.)Bi

olog

ical

proc

ess/

Mol

ecul

arfu

nctio

n

WT-

60’

vs.W

T-0’

sty1D

-60’

vs.s

ty1D

-0’

sty1D

-0’

vs.W

T-0’

sty1D

-60’

vs.W

T-60

’

1176

a2-

Hydr

oxy

acid

dehy

drog

enas

e(p

redi

cted

)SP

ACUN

K4.1

0–

1.5

–2

1.22

21.

76.

142

000

6.1

3670

0Am

ino

acid

met

abol

ism

(ser

ine

bios

ynth

esis

)13

1740

SRi

boso

mal

prot

ein

S12

SPCC

962.

04–

––

11.9

611

.22

4.45

1445

04.

4616

000

Prot

ein

synt

hesi

s(s

truct

ural

cons

titue

ntof

ribos

ome)

1727

60S

Ribo

som

alpr

otei

nL6

(frag

men

t?)

SPCC

622.

18–

1.69

–2

2.28

23.

785.

9294

7010

.27

2129

0Pr

otei

nsy

nthe

sis

(stru

ctur

alco

nstit

uent

ofrib

osom

e)65

56-

Phos

phog

luco

nate

dehy

dro-

gena

se,d

ecar

boxy

latin

gSP

BC66

0.16

1.1.

1.44

1.26

21.

132

1.48

22.

17.

4756

980

6.73

5422

0Pe

ntos

eph

osph

ate

path

way

and

hexo

sem

onop

hosp

hate

shun

t33

8Ac

etol

acta

tesy

ntha

seSP

BP35

G2.0

72.

2.1.

51.

262

1.17

21.

272

1.88

8.01

7085

09.

2873

370

Amin

oac

idm

etab

olis

m(v

alin

ean

dis

oleu

cine

bios

ynth

esis

;1st

step

)12

92Ac

etol

acta

tesy

ntha

sesm

all

subu

nitp

recu

rsor

(pre

dict

ed)

SPBC

14C8

.04

2.2.

1.6

21.

481.

71.

684.

224.

9733

080

5.94

3210

0Am

ino

acid

met

abol

ism

(val

ine

and

isol

euci

nebi

osyn

thes

is;1

stst

ep)

182

Ade1

,min

4,ph

osph

orib

osyl

amin

e-gl

ycin

elig

ase

SPBC

405.

016.

3.4.

132

1.26

21.

472

1.6

21.

864.

8880

480

5.37

8591

0Pu

rine

met

abol

ism

and

bios

ynth

esis

(2nd

step

)33

2Ad

e10,

bifu

nctio

nalp

urin

ebi

osyn

thes

ispr

otei

n(A

ICAR

trans

form

ylas

e/IM

Pcy

cloh

ydro

lase

)

SPCP

B16A

4.03

c2.

1.2.

3/3.

5.4.

101.

142

1.14

21.

252

1.62

7.05

7159

06.

3564

600

Purin

em

etab

olis

man

dbi

osyn

thes

is(9

than

d10

thst

eps)

1000

aAd

e6,m

in1,

phos

phor

ibos

ylam

ino-

imid

azol

eca

rbox

ylas

eSP

CC13

22.1

34.

1.1.

212

1.15

21.

353.

793.

227.

165

000

6.5

6000

0Pu

rine

met

abol

ism

and

bios

ynth

esis

(6th

step

)99

0bAd

e6,m

in1,

phos

phor

ibos

ylam

ino-

imid

azol

eca

rbox

ylas

eSP

CC13

22.1

34.

1.1.

212

1.56

–2

2.16

21.

297.

365

000

6.5

6000

0Pu

rine

met

abol

ism

and

bios

ynth

esis

(6th

step

)72

6Ad

enyl

osuc

cina

tesy

nthe

tase

SPAC

144.

036.

3.4.

42

1.04

1.11

21.

512

1.3

6.41

5459

06.

0548

250

Purin

em

etab

olis

man

dbi

osyn

thes

is(A

MP

bios

ynth

esis

;1st

step

),al

anin

ean

das

parta

tem

etab

olis

m11

28Al

do/k

eto

redu

ctas

eSP

BC8E

4.04

–1.

132

1.9

21.

642

3.52

7.61

3981

06.

6136

720

Carb

ohyd

rate

met

abol

ism

(oxi

dore

duct

ase

activ

ity)

1197

Aldo

/ket

ore

duct

ase

(role

infe

rred

from

hom

olog

y)SP

BC21

5.11

c–

8.41

21.

41.

792

6.59

7.02

3670

06.

4834

070

Unkn

own

(oxi

dore

duct

ase

activ

ity)

1232

aAl

do/k

eto

redu

ctas

eN

ADPH

-de

pend

ent(

pred

icte

d)SP

AC19

G12.

09–

3.05

–2

3.42

29.

637.

2835

470

6.33

3172

0Un

know

n(o

xido

redu

ctas

eac

tivity

)

1586

Bref

eldi

nA

resi

stan

cepr

otei

np2

0(fl

avod

oxin

-like

)a)SP

AC3C

7.14

c–

7.26

22.

8110

.22

21.

996.

6521

400

6.29

2189

0Un

know

n(ta

rget

ofpa

p1tra

nscr

iptio

nfa

ctor

and

conf

ers

bref

eldi

nA

resi

stan

ce)

1597

Bref

eldi

nA

resi

stan

cepr

otei

np2

0(fl

avod

oxin

-like

)a)SP

AC3C

7.14

c–

7.65

22.

3310

.66

21.

686.

720

530

6.29

2189

0Un

know

n(ta

rget

ofpa

p1tra

nscr

iptio

nfa

ctor

and

conf

ers

bref

eldi

nA

resi

stan

ce)


Proteomics 2006, 6, 2772–2796 Animal Proteomics 2783Tab

le3.

Co

nti

nu

ed

Spot

no.

Prot

ein

nam

eOR

Fna

me

E.C.

no.

Aver

age

fold

-cha

nge

pI,

gel

Mr

(gel

)pI (p

red)

Mr

(pre

d.)Bi

olog

ical

proc

ess/

Mol

ecul

arfu

nctio

n

WT-

60’

vs.W

T-0’

sty1D

-60’

vs.s

ty1D

-0’

sty1D

-0’

vs.W

T-0’

sty1D

-60’

vs.W

T-60

’

492a

Cata

lase

SPCC

757.

07c

1.11

.1.6

5.96

–2

1.74

215

.06

7.27

6387

06.

3958

470

Redo

xre

gula

tion,

prot

ects

cells

from

the

toxi

cef

fect

sof

hydr

ogen

pero

xide

)87

3aDi

hydr

oxya

ceto

neki

nase

1,da

k1SP

AC22

A12.

112.

7.1.

292

1.95

21.

931.

05–

765

500

6.2

6230

0Gl

ycer

olip

idm

etab

olis

m89

0DN

Ada

mag

ech

eckp

oint

prot

ein

Rad2

5(1

4–3-

3fa

mily

)SP

AC17

A2.1

3c–

1.08

21.

292

1.7

22.

374.

839

800

4.78

3057

0Ce

llcy

cle

regu

latio

n(c

ontro

lsDN

Ada

mag

ech

eckp

oint

befo

rem

itosi

s)93

8aEl

onga

tion

fact

or2

(frag

men

t?)

SPAC

513.

01–

21.

4–

1.79

2.38

5.78

4665

05.

9593

710

Prot

ein

synt

hesi

s(G

TP-b

indi

ng)

793

Elon

gatio

nfa

ctor

2(fr

agm

ent?

)SP

AC51

3.01

–2

1.25

1.23

3.23

4.98

6.1

5160

05.

9590

440

Prot

ein

synt

hesi

s(G

TP-b

indi

ng)

935c

Elon

gatio

nfa

ctor

2(fr

agm

ent?

)SP

AC51

3.01

–2

1.54

–2.

644.

276.

1246

720

5.95

9320

0Pr

otei

nsy

nthe

sis

(GTP

-bin

ding

)14

3El

onga

tion

fact

or2,

etf2

SPAC

513.

01c

–16

.55

––

210

.86

7.03

8451

06.

0293

800

Prot

ein

synt

hesi

s(G

TP-b

indi

ng)

675

Euka

ryot

ictra

nsla

tion

initi

atio

nfa

ctor

2bga

mm

asu

buni

tSP

AC4D

7.09

–1.

314.

282

1.28

2.56

6.68

5623

06.

2151

632

Prot

ein

synt

hesi

s

496

Gluc

ose-

6-ph

osph

ate

1-de

hydr

ogen

ase,

zwf1

a)SP

AC3A

12.1

81.

1.1.

492.

37–

21.

262

3.06

6.11

6359

05.

8357

620

Pent

ose

phos

phat

epa

thw

ay(1

stst

ep),

glut

athi

one

met

abol

ism

754a

Glut

amat

ede

hydr

ogen

ase

(NAD

P-sp

ecifi

c)SP

CC62

2.12

c1.

4.1.

4–

–2

3.21

24.

457.

6253

700

7.14

4915

8Am

ino

acid

met

abol

ism

(glu

tam

ate

met

abol

ism

)93

8bGl

utam

ine

synt

heta

se(g

luta

mat

e-am

mon

ialig

ase)

a)SP

AC23

H4.0

66.

3.1.

22

1.4

–1.

792.

385.

7846

650

5.89

4039

0Am

ino

acid

met

abol

ism

(con

vers

ion

ofgl

utam

ate

togl

utam

ine)

931

Glut

amin

esy

nthe

tase

(glu

tam

ate-

amm

onia

ligas

e)a)

SPAC

23H4

.06

6.3.

1.2

3.39

2.9

–1.

135.

8646

580

5.89

4039

2Am

ino

acid

met

abol

ism

(con

vers

ion

ofgl

utam

ate

togl

utam

ine)

941a

Glut

amin

esy

nthe

tase

(glu

tam

ate-

amm

onia

ligas

e)a)

SPAC

23H4

.06

6.3.

1.2

13.7

1–

21.

352

15.3

45.

9446

450

5.89

4039

0Am

ino

acid

met

abol

ism

(con

vers

ion

ofgl

utam

ate

togl

utam

ine)

947

Glut

amin

esy

nthe

tase

(glu

tam

ate-

amm

onia

ligas

e)a)

SPAC

23H4

.06

6.3.

1.2

21.

832

2.88

21.

272

26.

0646

580

5.89

4039

2Am

ino

acid

met

abol

ism

(con

vers

ion

ofgl

utam

ate

togl

utam

ine)

935b

Glut

amin

esy

nthe

tase

(glu

tam

ate-

amm

onia

ligas

e)a)

SPAC

23H4

.06

6.3.

1.2

21.

54–

2.64

4.27

6.12

4672

05.

8940

390

Amin

oac

idm

etab

olis

m(c

onve

rsio

nof

glut

amat

eto

glut

amin

e)12

10Gl

utat

hion

eS-

trans

fera

se2

SPCC

965.

07c

2.5.

1.18

3.58

21.

742.

622

2.38

6.05

2580

05.

9926

880

Redo

xre

gula

tion

and

deto

xific

atio

n12

03Gl

utat

hion

eS-

trans

fera

se2

SPCC

965.

07c

2.5.

1.18

2.08

––

21.

526.

226

200

5.99

2688

0Re

dox

regu

latio

nan

dde

toxi

ficat

ion

1204

Glut

athi

one

synt

heta

sela

rge

chai

n,gs

a1SP

AC3F

10.0

46.

3.2.

31.

91.

782

1.2

21.

276.

553

600

656

120

Redo

xre

gula

tion

(glu

tath

ione

bios

ynth

esis

;2n

dst

ep),

amin

oac

idm

etab

olis

m92

9aGl

ycer

ol-3

-pho

spha

tede

hydr

ogen

ase,

gpd1

a)SP

BC21

5.05

1.1.

1.8

21.

6–

1.62

3.21

3.76

4672

05.

2542

480

Glyc

erol

ipid

met

abol

ism

,stre

ssre

spon

se

836

Glyc

erol

-3-p

hosp

hate

dehy

drog

enas

e,gp

d1a)

SPBC

215.

051.

1.1.

81.

332

1.31

24.

092

7.14

4.23

5009

05.

2542

480

Glyc

erol

ipid

met

abol

ism

,stre

ssre

spon

se

849

Glyc

erol

-3-p

hosp

hate

dehy

drog

enas

e,gp

d1a)

SPBC

215.

051.

1.1.

81.

39–

23.

12

5.04

4.31

4987

05.

2542

480

Glyc

erol

ipid

met

abol

ism

,stre

ssre

spon

se

342a

GMP

synt

hase

(glu

tam

ine-

hydr

olys

ing)

(pre

dict

ed)g

ua2

SPAP

7G5.

02c

6.3.

5.2

1.3

21.

342

1.33

22.

326.

1260

290

6.12

6004

4Pu

rine

met

abol

ism

and

bios

ynth

esis

(GM

Pan

dgu

anin

ebi

osyn

thes

is)

1000

bGM

Psy

ntha

se(g

luta

min

e-hy

drol

ysin

g)(p

redi

cted

)SP

BPB2

B2.0

56.

3.5.

22

1.15

21.

353.

793.

227.

562

500

6.12

6020

0Pu

rine

met

abol

ism

and

bios

ynth

esis

(GM

Pan

dgu

anin

ebi

osyn

thes

is)



le3.

Co

nti

nu

ed

Spot

no.

Prot

ein

nam

eOR

Fna

me

E.C.

no.

Aver

age

fold

-cha

nge

pI,

gel

Mr

(gel

)pI (p

red)

Mr

(pre

d.)Bi

olog

ical

proc

ess/

Mol

ecul

arfu

nctio

n

WT-

60’

vs.W

T-0’

sty1D

-60’

vs.s

ty1D

-0’

sty1D

-0’

vs.W

T-0’

sty1D

-60’

vs.W

T-60

’

1230

GTP-

bind

ing

nucl

earp

rote

insp

i1(R

anGT

Pase

fam

ilym

embe

r)SP

BC12

89.0

3c–

–2

1.15

21.

432

1.62

4.75

3547

05.

424

150

Prot

ein

trans

port

(requ

ired

forp

rote

inim

-po

rtin

toth

enu

cleu

san

dfo

rRN

Aex

port)

1507

GTP-

bind

ing

nucl

earp

rote

insp

i1(R

anGT

Pase

fam

ilym

embe

r)SP

BC12

89.0

3c–

––

1.99

1.74

6.9

2500

07

2477

0Pr

otei

ntra

nspo

rt(re

quire

dfo

rpro

tein

im-

port

into

the

nucl

eus

and

forR

NA

expo

rt)39

2He

atsh

ock

cogn

ate

prot

ein,

hsc1

,sks

2a)SP

BC17

09.0

5–

21.

56–

1.31

2.13

5.02

6848

05.

8267

450

Mol

ecul

arch

aper

one,

stre

ssre

spon

se

331

Heat

shoc

kco

gnat

epr

otei

n,hs

c1,s

ks2a)

SPBC

1709

.05

–2

1.67

1.17

21.

241.

595.

3671

910

5.82

6745

0M

olec

ular

chap

eron

e,st

ress

resp

onse

328

Heat

shoc

kco

gnat

epr

otei

n,hs

c1,s

ks2a)

SPBC

1709

.05

–2

2.09

21.

212

1.5

1.15

5.61

7169

05.

8267

450

Mol

ecul

arch

aper

one,

stre

ssre

spon

se

314

Heat

shoc

kco

gnat

epr

otei

n,hs

c1,s

ks2a)

SPBC

1709

.05

––

–2

1.32

21.

65.

8972

440

5.82

6745

0M

olec

ular

chap

eron

e,st

ress

resp

onse

318

Heat

shoc

kco

gnat

epr

otei

n,hs

c1,s

ks2a)

SPBC

1709

.05

––

21.

062

1.45

21.

566.

1672

230

5.82

6745

0M

olec

ular

chap

eron

e,st

ress

resp

onse

354

Heat

shoc

kco

gnat

epr

otei

n,hs

c1,s

ks2a)

SPBC

1709

.05

–1.

54–

1.37

–7.

7169

600

5.82

6745

0M

olec

ular

chap

eron

e,st

ress

resp

onse

737

Heat

shoc

kco

gnat

epr

otei

n,hs

c1,s

ks2a)

(frag

men

t?)

SPBC

1709

.05

––

1.45

2.21

3.45

6.55

5443

05.

8267

450

Mol

ecul

arch

aper

one,

stre

ssre

spon

se

1320

Heat

shoc

kpr

otei

n16

SPBC

3E7.

02c

–2.

782.

88–

–5.

5113

900

5.72

1596

8M

olec

ular

chap

eron

e,st

ress

resp

onse

390

Heat

shoc

kpr

otei

n60

prec

urso

rSP

AC12

G12.

04–

1.11

–2

1.42

21.

574.

5668

070

5.76

6241

0M

olec

ular

chap

eron

e,st

ress

resp

onse

407

Heat

shoc

kpr

otei

n60

prec

urso

rSP

AC12

G12.

04–

21.

252

1.09

22.

082

1.82

4.68

6757

05.

7662

410

Mol

ecul

arch

aper

one,

stre

ssre

spon

se30

6He

atsh

ock

prot

ein

70fa

mily

mito

chon

dria

l,ss

c1,s

sp1a)

SPAC

664.

11–

21.

231.

042

1.66

21.

35.

4372

660

6.73

7316

0M

olec

ular

chap

eron

e,st

ress

resp

onse

287

Heat

shoc

kpr

otei

n70

fam

ilym

itoch

ondr

ial,

ssc1

,ssp

1a)SP

AC66

4.11

–2

1.13

21.

65–

21.

55.

6573

630

6.73

7316

0M

olec

ular

chap

eron

e,st

ress

resp

onse

668

Heat

shoc

kpr

otei

n70

fam

ily,s

sa2

(frag

men

t?)

SPCC

1739

.13

–2

1.57

1.34

3.21

6.74

5.55

5640

05.

1370

101

Mol

ecul

arch

aper

one,

stre

ssre

spon

se

716

Heat

shoc

kpr

otei

n70

fam

ily,s

sa2

(frag

men

t?)

SPCC

1739

.13

–2

1.4

1.49

24.

165.

9255

070

5.13

7010

1M

olec

ular

chap

eron

e,st

ress

resp

onse

324

Heat

shoc

kpr

otei

n70

fam

ily,s

sa2a)

SPCC

1739

.13

––

–1.

441.

544.

3372

800

5.13

7010

1M

olec

ular

chap

eron

e,st

ress

resp

onse

292

Heat

shoc

kpr

otei

n70

fam

ily,s

sa2a)

SPCC

1739

.13

–1.

062

1.21

21.

32

1.67

4.36

7280

05.

1370

101

Mol

ecul

arch

aper

one,

stre

ssre

spon

se27

6He

atsh

ock

prot

ein

70fa

mily

,ssa

2a)SP

CC17

39.1

3–

1.17

–2

1.37

21.

554.

4274

620

5.13

7010

1M

olec

ular

chap

eron

e,st

ress

resp

onse

299

Heat

shoc

kpr

otei

n70

fam

ily,s

sa2a)

SPCC

1739

.13

––

1.12

2.34

2.51

4.67

7280

05.

1370

101

Mol

ecul

arch

aper

one,

stre

ssre

spon

se98

3He

atsh

ock

prot

ein

70ho

mol

ogue

pss1

SPAC

110.

04c

–2

1.65

1.29

5.13

10.8

93.

8466

700

5.02

8055

4M

olec

ular

chap

eron

e,st

ress

resp

onse

625

Heat

shoc

kpr

otei

n70

hom

olog

ueps

s1SP

AC11

0.04

c–

21.

341.

312.

113.

685.

8668

010

5.02

8055

4M

olec

ular

chap

eron

e,st

ress

resp

onse

224

Heat

shoc

kpr

otei

n90

hom

olog

ue,

swo1

a)SP

AC92

6.04

c–

–2

1.32

21.

192

1.64

4.1

7906

04.

8980

710

Mol

ecul

arch

aper

one,

stre

ssre

spon

se(in

tera

cts

with

wee

1pr

otei

nki

nase

)



le3.

Co

nti

nu

ed

Spot

no.

Prot

ein

nam

eOR

Fna

me

E.C.

no.

Aver

age

fold

-cha

nge

pI,

gel

Mr

(gel

)pI (p

red)

Mr

(pre

d.)Bi

olog

ical

proc

ess/

Mol

ecul

arfu

nctio

n

WT-

60’

vs.W

T-0’

sty1D

-60’

vs.s

ty1D

-0’

sty1D

-0’

vs.W

T-0’

sty1D

-60’

vs.W

T-60

’

327

Heat

shoc

kpr

otei

n90

hom

olog

ue,

swo1

a)SP

AC92

6.04

c–

–1.

217.

9411

.05

4.22

7180

04.

8980

720

Mol

ecul

arch

aper

one,

stre

ssre

spon

se(in

tera

cts

with

wee

1pr

otei

nki

nase

)30

1He

atsh

ock

prot

ein

90ho

mol

ogue

,sw

o1a)

SPAC

926.

04c

––

–2.

282.

044.

2872

870

4.89

8071

0M

olec

ular

chap

eron

e,st

ress

resp

onse

(inte

ract

sw

ithw

ee1

prot

ein

kina

se)

679

Hydr

oxym

ethy

lglu

tary

l-CoA

synt

hase

SPAC

4F8.

14c

2.3.

3.10

21.

11.

852

1.26

1.61

6.03

5615

07.

0549

720

Ster

oid

bios

ynth

esis

,val

ine,

leuc

ine,

isol

euci

nede

grad

atio

n,ke

tone

body

synt

hesi

san

dde

grad

atio

n76

4Hy

poth

etic

alpr

otei

n(c

onse

rved

)SP

CC73

6.15

–1.

59–

22.

352

3.69

4.1

5315

05.

2439

952

Unkn

own

(resp

onds

tohe

at,p

rote

inki

nase

inhi

bito

rby

sim

ilarit

y)76

8Hy

poth

etic

alpr

otei

n(c

onse

rved

)SP

CC73

6.15

–1.

41–

23.

012

4.71

4.16

5307

05.

2439

952

Unkn

own

(resp

onds

tohe

at,p

rote

inki

nase

inhi

bito

rby

sim

ilarit

y)77

0Hy

poth

etic

alpr

otei

n(c

onse

rved

)SP

CC73

6.15

–1.

12–

23.

862

5.96

4.25

5284

05.

2439

805

Unkn

own

(resp

onds

tohe

at,p

rote

inki

nase

inhi

bito

rby

sim

ilarit

y)12

32b

Hypo

thet

ical

prot

ein

(con

serv

ed)

SPCC

777.

06c

–3.

05–

23.

422

9.63

7.28

3547

06.

234

240

Unkn

own

(pre

dict

edhy

drol

ase)

1176

bHy

poth

etic

alpr

otei

n44

.2k

(con

serv

ed)

SPAC

1002

.18

–1.

5–

21.

222

1.7

6.1

4200

06.

344

200

Unkn

own

941b

Hypo

thet

ical

serin

e-ric

hpr

otei

nSP

AC23

H3.1

5c–

13.7

1–

21.

352

15.3

45.

9446

450

5.86

3467

0Un

know

n(s

eque

nce

orph

an)

1131

Inor

gani

cpy

roph

osph

atas

eSP

AC3A

12.0

23.

6.1.

1–

1.21

1.3

1.58

4.22

3935

05.

232

680

Aero

bic

resp

iratio

n(d

ipho

spha

te1

H 2O

=2

Pi,i

nfer

red

from

hom

olog

y)91

6Le

u1,3

-isop

ropy

lmal

ate

dehy

drog

enas

eSP

BC1A

4.02

c1.

1.1.

85–

21.

8717

.04

9.95

5.01

4721

05.

3940

107

Amin

oac

idm

etab

olis

m(le

ucin

ebi

osyn

thes

is;3

rdst

ep)

164

Leu2

,3-is

opro

pylm

alat

ede

hydr

atas

ea)SP

AC9E

9.03

4.2.

1.33

21.

062

1.69

21.

882

36.

0982

530

5.75

8341

5Am

ino

acid

met

abol

ism

(leuc

ine

bios

ynth

esis

;2nd

step

)11

89M

alat

ede

hydr

ogen

ase,

mito

chon

dria

l(pr

edic

ted)

SPCC

306.

08c

1.1.

1.37

4.43

21.

493.

752

1.76

7.54

3719

08.

935

880

Pyru

vate

met

abol

ism

,citr

ate

cycl

e

935a

Met

al-d

epen

dent

hydr

olas

e(p

redi

cted

)SP

AC69

4.04

c–

23.

152

2.4

21.

41–

5.75

3810

05.

8137

020

Unkn

own

1016

Mito

chon

dria

lF1-

ATP

synt

hase

beta

subu

nit(

fragm

ent?

)SP

AC22

2.12

c3.

6.3.

142

1.56

–4.

337.

014.

9834

900

5.72

5689

8Ae

robi

cre

spira

tion

(ADP

1ph

osph

ate1

H1(O

ut)

=AT

P1

H2O1

H1(In

))75

4aN

ADP-

spec

ific

glut

amat

ede

hydr

ogen

ase

SPCC

622.

12c

1.4.

1.4

––

23.

212

4.45

7.62

5370

07.

1449

158

Amin

oac

idm

etab

olis

m(g

luta

mat

em

etab

olis

m)

1026

Orni

thin

eam

inot

rans

fera

se(fr

agm

ent?

)SP

BC21

C3.0

8c2.

6.1.

132

1.84

1.13

1.53

3.16

4.88

4300

05.

9448

670

Amin

oac

idm

etab

olis

m(a

rgin

ine

and

prol

ine

met

abol

ism

),ur

eacy

cle

654

Orni

thin

eam

inot

rans

fera

sea)

SPBC

21C3

.08c

2.6.

1.13

21.

7–

21.

76–

6.1

5715

05.

9448

670

Amin

oac

idm

etab

olis

m(a

rgin

ine

and

prol

ine

met

abol

ism

),ur

eacy

cle

731

Orni

thin

eam

inot

rans

fera

sea)

SPBC

21C3

.08c

2.6.

1.13

21.

54–

21.

892

1.34

6.36

5435

05.

9448

670

Amin

oac

idm

etab

olis

m(a

rgin

ine

and

prol

ine

met

abol

ism

),ur

eacy

cle



le3.

Co

nti

nu

ed

Spot

no.

Prot

ein

nam

eOR

Fna

me

E.C.

no.

Aver

age

fold

-cha

nge

pI,

gel

Mr

(gel

)pI (p

red)

Mr

(pre

d.)Bi

olog

ical

proc

ess/

Mol

ecul

arfu

nctio

n

WT-

60’

vs.W

T-0’

sty1D

-60’

vs.s

ty1D

-0’

sty1D

-0’

vs.W

T-0’

sty1D

-60’

vs.W

T-60

’

663

Orni

thin

eam

inot

rans

fera

sea)

SPBC

21C3

.08c

2.6.

1.13

21.

782

1.41

21.

922

1.52

6.37

5648

05.

9448

670

Amin

oac

idm

etab

olis

m(a

rgin

ine

and

prol

ine

met

abol

ism

),ur

eacy

cle

492b

Prot

ein

tyro

sine

phos

phat

ase,

pyp3

SPAC

11E3

.09

3.1.

3.48

5.96

–2

1.74

215

.06

7.27

6387

09.

3340

470

Sign

allin

g(re

gula

tion

ofth

ece

llcy

cle)

342b

Phos

phog

luco

mut

ase

prec

urso

r(p

redi

cted

)SP

BC32

F12.

105.

4.2.

21.

32

1.34

21.

332

2.32

6.09

6090

06.

0960

900

Glyc

olys

is,p

ento

seph

osph

ate

path

way

and

suga

rmet

abol

ism

762

Phos

phog

lyce

rate

dehy

drog

enas

e(p

redi

cted

)SP

CC36

4.07

1.1.

1.95

4.04

–2

1.31

23.

785.

9553

310

6.09

5119

0Am

ino

acid

met

abol

ism

(ser

ine

bios

ynth

esis

;1st

step

)21

3Po

lyad

enyl

ate-

bind

ing

prot

ein,

pab1

SPAC

57A7

.04c

–2

1.06

21.

312

1.99

22.

464.

7679

060

5.29

7175

0RN

Apr

oces

sing

(bin

dsth

epo

ly(A

)tai

lof

mRN

A)22

0Po

lyad

enyl

ate-

bind

ing

prot

ein,

pab1

SPAC

57A7

.04c

–2

1.21

21.

532

1.97

22.

484.

8778

590

5.29

7175

0RN

Apr

oces

sing

(bin

dsth

epo

ly(A

)tai

lof

mRN

A)11

03Po

lyad

enyl

ate-

bind

ing

prot

ein,

pab1

(frag

men

t?)

SPAC

57A7

.04c

––

1.19

2.73

2.91

4.84

3048

05.

2971

750

RNA

proc

essi

ng(b

inds

the

poly

(A)t

ailo

fm

RNA)

1080

bPr

otei

ndi

sulp

hide

isom

eras

epr

ecur

sor2

(pre

dict

ed)

SPAC

17H9

.14c

5.3.

4.1

21.

35–

–1.

534.

6841

380

5.54

4069

2M

olec

ular

chap

eron

e(c

atal

yses

rear

ran-

gem

ento

fpro

tein

disu

lphi

debo

nds)

1082

Prot

ein

disu

lphi

deis

omer

ase

prec

urso

r1(p

redi

cted

)SP

AC1F

5.02

5.3.

4.1

1.18

–2

1.45

21.

775.

241

380

5.54

5488

0M

olec

ular

chap

eron

e(c

atal

yses

rear

ran-

gem

ento

fpro

tein

disu

lphi

debo

nds)

1233

Pyrid

oxal

redu

ctas

e,pl

rSP

AC9E

9.11

1.1.

1.65

21.

192

1.27

2.52

2.36

7.62

3547

06.

7737

020

Coen

zym

ean

dpr

osth

etic

grou

pm

etab

olis

m(p

yrid

oxin

e(v

it.B6

)met

abol

ism

)11

27b

Quin

one

oxid

ored

ucta

se,m

ito-

chon

dria

lpep

tidas

e(p

redi

cted

)SP

BC16

A3.0

2c1.

6.5.

51.

442

1.92

–2

3.54

7.48

3981

06.

7138

510

Prot

ein

proc

essi

ng,m

ayal

soca

taly

seth

ere

duct

ion

ofce

rtain

quin

ones

330

Resi

dent

lum

inal

ERpr

otei

n,Gr

p78,

Bip1

SPAC

22A1

2.15

c–

1.2

2.06

21.

481.

164.

0871

800

4.83

7322

6M

olec

ular

chap

eron

e(re

quire

dfo

rfol

ding

and

asse

mbl

yof

mul

timer

icpr

otei

nco

mpl

exes

inth

eER

)12

36Ri

bose

5-ph

osph

ate

isom

eras

e(p

redi

cted

)SP

AC14

4.12

5.3.

1.6

–2

1.12

21.

572

1.75

5.79

3536

05.

8829

600

Pent

ose

phos

phat

epa

thw

ay

1224

bSe

c14

cyto

solic

fact

or,s

po20

SPAC

3H8.

10–

1.57

1.17

21.

212

1.62

7.33

3589

06.

6633

070

Prot

ein

trans

port,

glyc

erop

hosp

holip

idtra

nsfe

r,se

ptat

ion

and

spor

ulat

ion

241

Sti1

TPR

repe

atpr

otei

nSP

CC64

5.14

c–

1.13

21.

38–

21.

555.

0376

640

5.42

6601

0M

olec

ular

chap

eron

e,st

ress

resp

onse

(pre

dict

edro

lein

heat

shoc

kre

spon

seof

som

eHS

P70

gene

s)84

0Su

ccin

ate

sem

iald

ehyd

ede

hydr

ogen

ase

(pre

dict

ed)

SPAC

139.

051.

2.1.

166.

52–

––

6.47

6060

05.

7253

728

Amin

oac

idm

etab

olis

m(g

luta

mat

em

etab

olis

m)

769a

Suga

roxi

dore

duct

ase

SPAC

UNK4

.17

–2.

37–

21.

072

2.44

4.69

5276

05.

3145

820

Unkn

own

(oxi

dore

duct

ase

activ

ity)

38Su

lphi

tere

duct

ase

(pre

dict

ed)

SPCC

584.

01c

1.8.

1.2

21.

192

1.5

21.

532

1.92

4.91

9444

05.

3416

454

0Am

ino

acid

met

abol

ism

(cys

tein

ean

dm

ethi

onin

ebi

osyn

thes

is)

406

T-co

mpl

expr

otei

n1,

alph

asu

buni

tSP

BC12

D12.

03–

1.19

–2

1.52

21.

897.

0567

570

6.32

6035

0M

olec

ular

chap

eron

e(p

lays

aro

lein

fold

ing

ofac

tinan

dtu

bulin

)41

2T-

com

plex

prot

ein

1,ep

silo

nsu

buni

t(pr

edic

ted)

SPAC

1420

.02c

–1.

12

1.23

21.

662

2.25

6.16

6717

05.

8459

800

Mol

ecul

arch

aper

one

(pla

ysa

role

info

ldin

gof

actin

and

tubu

lin)



le3.

Co

nti

nu

ed

Spot

no.

Prot

ein

nam

eOR

Fna

me

E.C.

no.

Aver

age

fold

-cha

nge

pI,

gel

Mr

(gel

)pI (p

red)

Mr

(pre

d.)Bi

olog

ical

proc

ess/

Mol

ecul

arfu

nctio

n

WT-

60’

vs.W

T-0’

sty1D

-60’

vs.s

ty1D

-0’

sty1D

-0’

vs.W

T-0’

sty1D

-60’

vs.W

T-60

’

417

T-co

mpl

expr

otei

n1,

zeta

subu

nit

SPBC

646.

11–

1.22

21.

142

1.5

22.

086.

2566

970

5.9

5885

0M

olec

ular

chap

eron

e(p

lays

aro

lein

fol-

ding

ofac

tinan

dtu

bulin

)15

82Th

iore

doxi

npe

roxi

dase

a)SP

CC57

6.03

c–

1.65

1.11

21.

812

2.68

4.34

2150

05.

3721

290

Redo

xre

gula

tion

(per

oxid

ase

activ

ityre

-qu

ired

fort

hetra

nscr

iptio

nalr

espo

nse

toox

idat

ive

stre

ss)

1576

Thio

redo

xin

pero

xida

sea)

SPCC

576.

03c

–2.

172

1.67

21.

242

4.48

4.64

2191

05.

3721

290

Redo

xre

gula

tion

(per

oxid

ase

activ

ityre

-qu

ired

fort

hetra

nscr

iptio

nalr

espo

nse

toox

idat

ive

stre

ss)

1172

Thio

redo

xin

redu

ctas

eSP

BC3F

6.03

1.6.

4.5

2.48

2.49

–2

1.29

4.59

3780

05.

1934

790

Redo

xre

gula

tion

(rege

nera

tion

ofre

duce

dth

iore

doxi

n)11

74Th

iore

doxi

nre

duct

ase

SPBC

3F6.

031.

6.4.

53.

892

1.18

–2

4.75

4.74

3780

05.

1934

790

Redo

xre

gula

tion

(rege

nera

tion

ofre

duce

dth

iore

doxi

n)93

9bTr

ansa

ldol

ase

SPCC

1020

.06c

––

––

22.

395.

3637

965

5.81

3702

0Pe

ntos

eph

osph

ate

path

way

(non

-oxi

dativ

epa

rt)24

4Tr

ansk

etol

ase

(pre

dict

ed)

SPBC

2G5.

05–

1.16

1.62

21.

091.

296.

8876

410

6.33

7576

2Pe

ntos

eph

osph

ate

path

way

320

Vacu

olar

ATP

synt

hase

cata

lytic

subu

nita

SPAC

343.

053.

6.3.

141.

172

1.17

21.

432

1.97

5.95

7223

05.

869

200

ATP

synt

hesi

s,ac

idifi

catio

nof

intra

cellu

lar

com

partm

ents

431

Vacu

olar

ATP

synt

hase

subu

nitb

SPAC

17A2

.03c

3.6.

3.14

1.3

21.

362

1.58

22.

86.

6366

380

5.19

5586

0AT

Psy

nthe

sis,

acid

ifica

tion

ofin

trace

llula

rco

mpa

rtmen

ts12

12Vi

p1pr

otei

nSP

AC10

F6.0

6–

1.49

–2

1.55

21.

964.

330

000

5.54

2750

0Un

know

n(p

utat

ive

RNA

bind

ing

prot

ein)

1215

Vip1

prot

ein

SPAC

10F6

.06

––

–2

2.48

23.

394.

7936

320

5.54

2750

0Un

know

n(p

utat

ive

RNA

bind

ing

prot

ein)

1068

bZi

nc-b

indi

ngde

hydr

ogen

ase

SPAC

2E1P

3.01

–2.

31.

151.

712

1.17

7.15

4187

06.

3337

960

Unkn

own

(zin

c-de

pend

enta

lcoh

olde

hy-

drog

enas

eac

tivity

)



Figure 6. Effect of H2O2 on thegrowth of SPAC23H3.15cD SPA-CUNK4.17D sty1D and atf1Ddeletions mutants comparedwith WT. Cells were seriallydiluted and plated on YE mediawith and without H2O2 (at0.5 mM and 1 mM) and grownfor 4 days at 307C.

up-regulated in sty1D (Table 3). This suggests that Sty1p maynormally inhibit proteolytic cleavage of Pab1p and therebyalter translation initiation to affect protein expression. Sev-eral coregulated proteins could also be grouped according totheir inclusion in multi-subunit complexes. For example,subunits E1 and E2 of pyruvate dehydrogenase were co-regulated (Table 2), and may affect pyruvate levels and hencethe metabolic pathways that utilize pyruvate and acetyl CoA.Similarly, subunits a and b of the vacuolar ATP synthasewere both repressed in sty1D cells (Table 3), suggesting thatSty1p may regulate vacuolar acidification and protein pro-cessing. Three subunits of the chaperonin-containing T-complex were also down-regulated in the sty1D mutant,again implicating Sty1p signalling in the regulation of cel-lular chaperone activity. However, coregulation was not ageneral observation, as in the case of acetolactate synthase(involved in the first step of valine, leucine and isoleucinesynthesis), where the large and small subunits were oppo-sitely regulated (Table 3). Thus, further functional studiesare required to assess the consequence of such subunitchanges on complex activity.

3.4 Correlation of changes in protein and mRNA

abundance

An important resource in interpretation of the protein datahas been the publication of the complete fission yeast genomeand the subsequent publication of a global investigation of thetranscriptional responses of S. pombe to a number of environ-mental stresses [1]. This allowed the parallel comparison ofour quantitative proteomics data with quantitative mRNA datafor the same set of genes under the same conditions. Figure 7displays the overall correlations between published micro-array data and proteomic data for the four comparisons. Theoverall level of correlation was low (e.g. R2 = 0.2 for WT-60’ vs.WT-0’) in agreement with previously published data [53, 54]and did not change when only singly identified isoforms wereconsidered. Of particular interest was the very low correlationswhen comparing sty1D-60’ versus sty1D-0’ (R2 = 0.0033,Fig. 7B) and untreated sty1D versus WT (R2 = 0.045, Fig. 7C).This suggests a significant increase in PTMs (possibly irre-versible oxidation of cysteinyl thiols) and that loss of Sty1p hasa more profound effect on post-transcriptional events than ongene expression per se. The correlation between sty1D and WT

cells under stress (R2 = 0.1768, Fig. 7D) is harder to explain asit would be expected that this correlation would be the lowestgiven the above assumptions. One possibility is that Sty1p-in-dependent, H2O2-inducible gene transcription componentincreases the correlation.

The mRNA analysis showed 44 gene products wererepressed in the absence of Sty1p, while the protein analysisidentified 51 repressed gene products with eight matching tothe mRNA data (Table 4). In most cases, the same direction-ality of mRNA and protein regulation was observed for thisgroup, identifying them as Sty1p-dependent gene targets.Conversely, 95 genes and 57 gene products were de-repressedby loss of Sty1p, with eight matching between the data sets,though with less correlation between transcription and pro-tein expression. Of the 56 peroxide-specific and 12 super-induced genes identified by microarray analysis, only eightgene products were identified by proteomic analysis (Table 4).All of these except a putative aminotransferase(SPAC56E4.03) displayed Sty1p-dependent induction of theprotein that agreed with the mRNA data, although changes inthioredoxin reductase were isoform-dependent. The inductionof thioredoxin reductase seems logical given its role in theregeneration of reduced thioredoxin. However, possible rolesfor brefeldin A-resistance protein, 2-hydroxy acid dehy-drogenase and protein-tyrosine phosphatase/Pyp3p are lessclear. In the transcriptional analysis, 140 genes induced by anumber of different environmental stresses were termed coreenvironmental stress response (CESR) genes [1]. Within thisgroup, 16 were identified by 2-D-DIGE/MS and all were up-regulated with H2O2 in the WT but not sty1D cells, exceptpyridoxal reductase, which was overexpressed in sty1D(Table 4). This group included seven enzymes involved in car-bohydrate metabolism and generation of NADPH reducingequivalents (glucose-6-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, malate dehydrogenase, succinatesemialdehyde dehydrogenase and three putative oxido-reductases), the redox enzymes catalase and thioredoxin per-oxidase and five proteins of unknown function, including thetwo that were characterized by assessing the H2O2 sensitivityof their deletion mutants (see above).

Hsp16 was the only Hsp family member identifiedwhere an increase in mRNA levels correlated with increasedprotein expression, identifying it as a stress-inducible gene.Importantly, expression of most S. pombe Hsp family genes



Table 4. Comparison of protein and mRNA ratios. Table shows the fold-change of protein isoform and mRNA across the four experimentalconditions for genes found in [1] that were; H2O2-specific genes, CESR genes super-induced with H2O2, genes that required Sty1pfor basal level expression, genes that were de-repressed in unstressed sty1D, induced CESR genes, repressed CESR genes

ORF Name Name mRNA ratio Protein ratio Function/Pathway

WT-60’vs.WT-0’

sty1D-60’vs.sty1D-0’

sty1D-0’vs.WT-0’


WT-60’vs.WT-0’



Sty1D-60’vs.WT-60’

H2O2-specific genes

SPAC3C7.14c Brefeldin A resistance proteinp20 (flavodoxin-like)

7.07 2.36 1.05 0.35 7.26 0.36 10.22 0.50 Unknown

SPAC3C7.14c Brefeldin A resistance proteinp20 (flavodoxin-like)

7.07 2.36 1.05 0.35 7.65 0.43 10.66 0.60 Unknown

SPAC56E4.03 Aminotransferase (putative) 2.50 1.81 1.28 0.92 0.51 0.52 1.05 – Unknown(amino acid metabolism)

SPACUNK4.10 2-hydroxy acid dehydrogenase(predicted)

4.34 1.07 0.91 0.22 1.50 – 0.82 0.59 Amino acid metabolism(serine biosynthesis)

SPBC3F6.03 Thioredoxin reductase 16.43 2.63 1.09 0.17 2.48 2.49 – 0.78 Redox regulationSPBC3F6.03 Thioredoxin reductase 16.43 2.63 1.09 0.17 3.89 0.85 – 0.21 Redox regulationSPAC11E3.09 Protein tyrosine phosphatase,

pyp33.60 2.16 0.78 0.93 5.96 – 0.57 0.07 Signalling

(regulation of cell cycle)

CESR genes super-induced with H2O2

SPAC19G12.09 Aldo/keto reductaseNADPH-dependent (pred.)

15.18 0.78 0.37 0.02 3.05 – 0.29 0.10 Unknown(oxidoreductase activity)

SPBC215.11c Aldo/keto reductase(role inferred from homology)

17.69 1.89 0.63 0.07 8.41 0.71 1.79 0.15 Unknown(oxidoreductase activity)

SPAC139.05 Succinate semialdehydedehydrogenase

128.57 – – 0.01 6.52 – – – Amino acid metabolism(glutamate metabolism)

Genes requiring Sty1p for basal expression in unstressed cells

SPCC757.07c Catalase 37.45 2.50 0.20 0.01 5.96 – 0.57 0.07 Redox regulationSPBC215.05 Glycerol-3-phosphate

dehydrogenase, gpd12.24 0.50 0.16 0.04 1.33 0.76 0.24 0.14 Glycerolipid metabolism

SPBC215.05 Glycerol-3-phosphatedehydrogenase, gpd1

2.24 0.50 0.16 0.04 1.39 – 0.32 0.20 Glycerolipid metabolism



SPCC794.12c Malate oxidoreductase(NAD-dependent)

1.11 1.05 0.41 0.39 – 0.85 0.31 0.26 Pyruvate metabolism


1.11 1.05 0.41 0.39 0.87 0.57 0.64 0.42 Pyruvate metabolism


1.11 1.05 0.41 0.39 0.63 – 1.90 3.12 Pyruvate metabolism

SPAC11E3.09 Protein tyrosine phosphatase,pyp3

1.81 2.16 0.78 0.93 5.96 – 0.57 0.07 Signalling (regulation of cellcycle)

SPBC16A3.02c Quinone oxidoreductase 10.54 0.81 0.48 0.04 1.44 0.52 – 0.28 Protein processingSPCC622.12c Glutamate dehydrogenase

(NADP-specific)2.09 0.94 0.46 0.21 – – 0.31 0.22 Amino acid metabolism

(glutamate metabolism)SPCC736.15 Hypothetical protein

(conserved)4.29 0.98 0.44 0.10 1.59 – 0.43 0.27 Unknown

SPCC736.15 Hypothetical protein(conserved)

4.29 0.98 0.44 0.10 1.41 – 0.33 0.21 Unknown


4.29 0.98 0.44 0.10 1.12 – 0.26 0.17 Glycolysis, pyruvate metabolism

SPAC10F6.06 Vip1 protein 3.32 1.56 0.42 0.20 1.49 – 0.65 0.51 Unknown (putative RNA bindingprotein)

SPAC10F6.06 Vip1 protein 3.32 1.56 0.42 0.20 – – 0.40 0.29 Unknown (putative RNA bindingprotein)



Table 4. Continued


WT-60’vs.WT-0’




WT-60’vs.WT-0’




Genes that are de-repressed in unstressed Sty1p-deleted cells

SPBC16H5.02 Pfk1, 6-phosphofructokinase(predicted)

0.54 0.06 7.84 0.87 – – 1.32 1.54 Glycolysis (key control step)

SPBC16H5.02 Pfk1, 6-phosphofructokinase(predicted)

0.54 0.06 7.84 0.87 0.86 0.87 1.51 1.52 Glycolysis (key control step)

SPCC584.01c Sulphite reductase (predicted) 0.31 0.08 6.75 1.83 0.84 0.67 0.65 0.52 Amino acid metabolism (cys andmet biosynthesis)

SPCC965.07c Glutathione S-transferase II 6.23 0.80 2.91 0.38 2.08 – – 0.66 Redox regulation anddetoxification

SPCC965.07c Glutathione S-transferase II 6.23 0.80 2.91 0.38 3.58 0.57 2.62 0.42 Redox regulation anddetoxification

SPBPB2B2.05 GMP synthase (predicted) 0.25 0.72 2.35 6.68 0.87 0.74 3.79 3.22 Purine metabolism andbiosynthesis

SPBC3E7.02c Heat shock protein 16 2.63 1.91 2.24 1.63 2.78 2.88 – – Molecular chaperone, stressresponse

SPBC19C7.06 Prolyl-tRNA synthetase 0.28 0.32 2.16 2.42 – 1.92 0.86 – Protein synthesisSPBC21C3.08c Ornithine aminotransferase 0.49 1.50 2.13 6.55 0.59 – 0.57 – Amino acid metabolism (arg and

pro metabolism)SPBC21C3.08c Ornithine aminotransferase 0.49 1.50 2.13 6.55 0.56 0.71 0.52 0.66 Amino acid metabolism (arg and

pro metabolism)SPBC21C3.08c Ornithine aminotransferase 0.49 1.50 2.13 6.55 0.65 – 0.53 0.75 Amino acid metabolism (arg and

pro metabolism)SPBC21C3.08c Ornithine aminotransferase

(fragment?)0.49 1.50 2.13 6.55 0.54 1.13 1.53 3.16 Amino acid metabolism (arg and

pro metabolism)SPBC215.08c Carbamoyl-phosphate

synthase, arg41.67 0.29 2.03 0.36 – 0.83 0.75 0.61 Amino acid metabolism

(arginine biosynthesis)

Induced CESR genes

SPAC19G12.09 Aldo/keto reductaseNADPH-dependent (pred.)

15.18 0.78 0.37 0.02 3.05 – 0.29 0.10 Unknown(oxidoreductase activity)

SPACUNK4.17 Sugar oxidoreductase 20.97 1.20 0.88 0.05 2.37 – 0.93 0.41 Unknown(oxidoreductase activity)

SPBC215.11c Aldo/keto reductase (roleinferred from homology)

17.69 1.89 0.63 0.07 8.41 0.71 1.79 0.15 Unknown(oxidoreductase activity)

SPCC306.08c Malate dehydrogenase,mitochondrial (predicted)

3.63 2.17 0.84 0.50 4.43 0.67 3.75 0.57 Pyruvate metabolism, citratecycle

SPAC3A12.18 Glucose-6-phosphate1-dehydrogenase, zwf1

6.32 1.24 0.78 0.15 2.37 – 0.79 0.33 Pentose phosphate pathway,GSH metabolism


2.24 0.50 0.16 0.04 1.33 0.76 0.24 0.14 Glycerolipid metabolism





SPAC139.05 Succinate semialdehydedehydrogenase

128.57 – – 0.01 6.52 – – – Amino acid metabolism(glutamate metabolism)

SPCC757.07c Catalase 37.45 2.50 0.20 0.01 5.96 – 0.57 0.07 Redox regulationSPCC576.03c Thioredoxin peroxidase 6.65 1.78 1.30 0.35 2.17 0.60 0.81 0.22 Redox regulationSPCC576.03c Thioredoxin peroxidase 6.65 1.78 1.30 0.35 1.65 1.11 0.55 0.37 Redox regulationSPBC3E7.02c Heat shock protein 16 2.63 1.91 2.24 1.63 2.78 2.88 – – Molecular chaperone, stress

responseSPAC9E9.11 Pyridoxal reductase, plr 3.75 1.04 1.66 0.46 0.84 0.79 2.52 2.36 Coenzyme and prosthetic group

metabolism



Table 4. Continued


WT-60’vs.WT-0’




WT-60’vs.WT-0’




SPBC16A3.02c Quinone oxidoreductase 10.54 0.81 0.48 0.04 1.44 0.52 – 0.28 Protein processingSPAC2E1P3.01 Zinc-binding dehydrogenase 8.85 0.79 0.84 0.07 2.30 1.15 1.71 0.85 Unknown (alcohol

dehydrogenase activity)SPAC10F6.06 Vip1 protein 3.32 1.56 0.42 0.20 1.49 – 0.65 0.51 Unknown (putative RNA binding

protein)SPAC10F6.06 Vip1 protein 3.32 1.56 0.42 0.20 – – 0.40 0.29 Unknown (putative RNA binding

protein)SPAC23H3.15c Hypothetical serine-rich protein 95.35 1.42 0.91 0.01 13.71 – 0.74 0.07 Unknown (sequence orphan)SPCC736.15 Hypothetical protein

(conserved)4.29 0.98 0.44 0.10 1.59 – 0.43 0.27 Unknown


4.29 0.98 0.44 0.10 1.41 – 0.33 0.21 Unknown


4.29 0.98 0.44 0.10 1.12 – 0.26 0.17 Glycolysis, pyruvate metabolism

Repressed CESR genes

SPCC285.03 DEAD/DEAH box 3’-5’DNA/RNA helicase (pred.)

0.89 0.88 1.03 1.03 – 0.75 0.63 0.47 RNA processing

SPAC23H4.06 Glutamine synthetase 0.14 1.57 1.15 12.63 3.39 2.90 – 1.13 Amino acid metabolism(conversion of glu to gln)

SPAC23H4.06 Glutamine synthetase 0.14 1.57 1.15 12.63 0.55 0.35 0.79 0.50 Amino acid metabolism(conversion of glu to gln)

SPAC23H4.06 Glutamine synthetase 0.14 1.57 1.15 12.63 0.65 – 2.64 4.27 Amino acid metabolism(conversion of glu to gln)



SPBC21C3.08c Ornithine aminotransferase 0.49 1.50 2.13 6.55 0.59 – 0.57 – Amino acid metabolism (arg andpro metabolism)

SPBC21C3.08c Ornithine aminotransferase 0.49 1.50 2.13 6.55 0.56 0.71 0.52 0.66 Amino acid metabolism (arg andpro metabolism)

SPBC21C3.08c Ornithine aminotransferase 0.49 1.50 2.13 6.55 0.65 – 0.53 0.75 Amino acid metabolism (arg andpro metabolism)

SPBC21C3.08c Ornithine aminotransferase(fragment?)

0.49 1.50 2.13 6.55 0.54 1.13 1.53 3.16 Amino acid metabolism (arg andpro metabolism)

SPBC25B2.09c Arginyl-tRNA synthetase(predicted)

0.34 0.67 1.21 2.43 1.24 – – 0.64 Protein synthesis

SPBC8D2.18c Adenosylhomocysteinase(predicted)

0.39 1.53 0.94 3.68 1.20 – 0.78 0.63 Amino acid metabolism (metmetabolism)

was transcriptionally down-regulated by H2O2 [1]. Of the 106down-regulated CESR genes, 6 appeared in the protein listand all were down-regulated in sty1D cells with the exceptionof ornithine aminotransferase (up-regulated) and glutaminesynthetase (where four isoforms displayed different expres-sion). In some instances where two proteins were identifiedin the same spot, examination of the mRNA data was used topredict which of the proteins was likely to be regulated. Forexample, hypothetical serine-rich protein (SPAC23H3.15c)and an isoform of glutamine synthetase were found in the

same spot (spot 941) which was strongly induced by H2O2 ina Sty1p-dependent manner. Since the expression of theSPAC23H3.15c mRNA was also strongly induced, the up-regulation of this protein is likely to account for the increasedspot intensity. Catalase and protein tyrosine phosphatase/Pyp3p were found to comigrate (spot 492) and the abun-dance of this spot was increased by H2O2 in a Sty1p-depend-ent manner. The mRNAs of both genes also displayed Sty1p-dependent H2O2 induction (Table 4) and so the observedchange in spot abundance is likely to be an additive effect.



Figure 7. Global correlations ofmRNA and protein data. Log10values of the mRNA ratios for allidentified gene products [1]were plotted against the log10protein ratios from the prote-omic analysis for the fourexperimental comparisons; (A)WT-60’ vs. WT-0’, (B) sty1D-60’vs. sty1D-0’, (C) sty1D-60’ vs.WT-0’ and (D) sty1D-60’ vs. WT-60’ and linear regression coeffi-cients (R2) for the data deter-mined.

3.5 Metabolomic analysis

A metabolite analysis was also conducted on the same sam-ple sets using 1H-NMR. Samples from five biological repli-cates were prepared for identical analyses with an internalstandard for calibration and quantitation of metabolitepeaks. In total 31 individual peaks were quantified and ofthese 29 have been previously assigned to a specific cellularmetabolite. Both Sty1p- and H2O2-specific changes wereapparent (Fig. 8). Loss of Sty1p resulted in a significant(p,0.05) reduction of the amino acids leucine, isoleucine,valine, alanine, glutamate, glutamine, tyrosine, histidine,phenylalanine and of the metabolites lactate, U1 (uni-dentified peak), glycerophosphocholine (GPC), trimethyl-amine-N-oxide (TMAO), glycerol and UTP 1 UDP. Con-versely, betaine (involved in glycine metabolism), glycine, b-D-glucose, orotic acid (involved in pyrimidine metabolism)and the ATP 1 ADP pool were increased in unstressed sty1Dcells (Fig. 8A). Peroxide treatment of WT cells resulted in theinduction of acetate, glutamate, lysine, choline, GPC,TMAO, betaine, glycine, glycerol, U2, UTP 1 UDP and ino-sine, and all of these, with the exception of inosine, displayeda lower level of induction or even repression in sty1D(Fig. 8B). Of the metabolites repressed in the WT, leucine,valine and U1 were actually induced in sty1D. The moststriking difference was a six-fold increase in b-D-glucose inthe sty1D cells, but not the WT, in response to H2O2. Togetherthese data show that the Sty1p pathway is a critical regulatorof multiple, diverse metabolic pathways, including aminoacid biosynthesis and metabolism, and is required for thenormal response to oxidative stress. Repression of amino

acids in sty1D may be due to a slower rate of cellular metab-olism, resulting in reduced protein synthesis (lowered levelsof branched amino acids) and membrane turnover(decreased GPC level). With a slower rate of cell metabolism,less energy will be required by these cells, which in turncould lead to reduced glycolysis (reduced level of lactate andelevated glucose level) and amino acid metabolism.

The changing metabolite profiles were compared withthe protein data to identify correlations between the regula-tion of specific enzymes and their substrates or products. Forexample, higher levels of glycine in sty1D may be explainedby the lower expression of the major glycine-degrading en-zyme glycine dehydrogenase (SPAC13G6.06c), which may inturn affect betaine levels and purine metabolism, since gly-cine is essential for purine synthesis. The data also suggestthat an H2O2- and Sty1p-dependent conversion of glutamineto glutamate, possibly involving the regulated expression ofglutamine synthetase (SPAC23H4.06), glutamate dehy-drogenase (SPCC622.12c) and/or succinate semialdehydedehydrogenase (SPAC139.05). Induction of the latter mayalso produce the observed reduction in succinate (Fig. 8B).Sty1p-dependent induction of D-1-pyroline-5-carboxylatedehydrogenase (SPBC24C6.04) could also raise glutamatelevels by conversion from proline, whilst overexpression ofthe glutamine-hydrolysing enzyme GMP synthase(SPBPB2B2.05) in sty1D would reduce glutamine and alterpurine metabolism (Table 3). Enzymatic synthesis of GSHoccurs from the component amino acids (glutamate, cyste-ine, and glycine) via the sequential action of two ATP-de-pendent cytosolic enzymes, so it may be that the observedincreases in glutamate and glycine levels enable increased de



Figure 8. Multiple metaboliteanalysis using high-resolution1H NMR spectroscopy. Five bio-logical sample repeats wereprepared and analysed accord-ing to ‘Section 2’. Percentagechanges in all metabolites areshown for; (A) sty1D vs. WTunder conditions of no stressand stress and (B) H2O2 stressvs. no stress for each strain.Data are expressed as themean 6 SEM with * indicatingsignificant changes (p , 0.05).Peaks labelled U1 and U2 couldnot be assigned.

novo synthesis of GSH for the provision of reducingpotential. The Sty1p-dependent increase in glycerol follow-ing stress is also interesting, since this metabolite isknown to play a cytoprotective role, and its induction maybe mediated via up-regulation of glycerol-3-phosphatedehydrogenase/Gpd1 (SPBC215.05) or alcohol dehy-drogenase (SPAC3A11.12c). In turn, higher glycerol levelsmay increase GPC generation and alter membrane turn-over. Like glycerol, TMAO is thought to act as a chemical

chaperone to protect proteins from misfolding and so itsup-regulation following H2O2 exposure (in a Sty1-depend-ent manner) may be an additional mechanism to protectagainst oxidative protein damage. Glycerol, TMAO, GPC,choline and betaine are also known osmoprotective metab-olites, and their induction suggests that some level ofcross-protection may exist between the peroxide andosmotic stress response, a theory which warrants furtherinvestigation.



Figure 9. Schematic representations ofmetabolic responses to H2O2 in (A) WTand (B) sty1D. Arrows between path-ways indicate known connections takenfrom the Kyoto Encyclopaedia of Genesand Genomes (KEGG) Pathways Data-base. Vertical arrows indicate a predic-tion of the up- or down-regulation of apathway based upon how protein and/ormetabolite levels change in response tostress. Stress response of the WT glyco-lytic pathway is more complex, sincethere was down-regulation of theenzymes in the lower half of pathwayand up-regulation of the enzymes in theupper part of the pathway (see text).Metabolic pathways in bold italic typeindicate that the predicted direction ofregulation of this pathway is supportedby protein data only whilst those in nor-mal type face indicate that the directionof regulation is supported by bothmetabolite and protein data.

4 Concluding remarks

In this study, we have identified a large number of S. pombeprotein isoforms that display H2O2-dependent changes inabundance and have related these changes to alterations inthe mRNA and metabolic profiles of cells treated underidentical conditions. This work shows that a diverse range ofcellular processes are affected by oxidative stress and furtherdemonstrates the role of the Sty1p signalling pathway inregulating some of these processes. Although a number ofthe recorded protein changes correlated well with alteredgene expression (Table 4), there was a low overall correlationbetween protein and mRNA expression in agreement withsome previous studies [53, 54], but not others, where higher

correlations were observed for specific subsets of genes [55,56]. Our work thus highlights the complexity of the cellularresponse to stress and indicates that many levels of regula-tion are at play. Indeed, a large number of proteins appearedto be post-translationally altered through proteolysis, phos-phorylation and/or oxidation with no correlative change intheir mRNA expressions. However, mRNA stability or theefficiency of translation may also be regulated by stress [57]and therefore may contribute to the low correlation observed.This emphasizes the requirement for more detailed prote-omic analyses if we are to fully understand the molecularmechanisms involved in responses to stress and other sti-muli. Moreover, given the relatively poor sensitivity and cov-erage of current proteomic methodologies, it is likely that



there are many more low-abundance targets to be identified,although it is important to note that 2-D-DIGE does provide arobust, quantitative method for global expression profiling.

When interpreted alone, each experimental methodidentifies putative targets of the oxidative stress response andspecifically the Sty1p-responsive elements of numerousmetabolic pathways. However, the real value of the data canonly be discerned when data sets are combined and theresults viewed globally. Such a systems approach allows sig-nificantly more detail to be proposed regarding how variouscellular processes are affected by oxidative stress and Sty1psignalling and how regulation is achieved. It is clear that thestress response is global and has profound effects on multi-ple, diverse metabolic pathways and that the Sty1p stress-activated protein kinase is a critical modulator of these eventsbasally, and in particular, during the stress response. Itinvolves a number of changes including alterations in pro-tein and amino acid synthesis, a switch in energy productionfrom glycolysis to the pentose phosphate pathway for thegeneration of NADPH and the induction of stress-specificantioxidant enzymes and cytoprotectants that would act toreduce cellular ROS, protect against molecular damage andreverse oxidative protein modification. This is in generalagreement with the conclusions of a previous proteomicanalysis of the H2O2 response in the budding yeast S. cerevi-siae [49], and indeed, there was considerable overlap in theprotein changes identified between this study and ours, par-ticularly of enzymes involved in carbohydrate and aminoacid metabolism. Finally, whilst WT cells elicit a robust re-sponse that is required for their survival, the sty1D responseis muted and appears to involve shutting down of proteinsynthesis, a number of metabolic pathways and energy pro-duction possibly in preparation for entering a dormant phaseor prior to cell death (Fig. 9).

We would like to thank Mr. Richard Jacob (UCL) for soft-ware writing and support for MS data analysis and the EMFBiological Research Trust for funding. We would also like toacknowledge The Medical Biomics Centre at St George’s, London,for the use of their 600 MHz NMR Spectrometer.

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A parallel proteomic and metabolomic analysis of the hydrogen peroxide- and Sty1p-dependent stress response inSchizosaccharomyces pombe

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