Proteomic Analysis of the Oxidative Stress Response in Kluyveromyces lactis and Effect of Glutathione Reductase Depletion A. Garcı´a-Leiro, M. E. Cerda´n, and M. I. Gonza´lez-Siso* Department of Molecular and Cell Biology, University of A Corun ˜ a, Campus da Zapateira s/n, 15071- A Corun ˜ a, Spain Received November 25, 2009
Yeasts are unicellular eukaryotes that provide useful models for studying the oxidative stress (OS) response. Most investigations to date have been performed on the fermentative Saccharomyces cerevisiae. The respiratory Kluyveromyces lactis is emerging as an alternative model. Our previous studies showed that glutathione reductase (Glr1) is an interesting point of difference in the OS response between the two yeasts. In the present study, using extensive proteomic analyses, the response to H2O2 and its relationship to Glr1 were investigated in wild-type and glr1-deletion mutant K. lactis strains. We identified 46 proteins that showed modified expression after H2O2 addition and 42 for which the change was Glr1-dependent. As expected, these proteins include a variety of antioxidant enzymes, chaperones, and oxidoreductases related to defense against OS and damage repair. They also include a number of proteins necessary for energy production and carbohydrate and amino acid metabolism. H2O2 addition causes down-regulation of enzymes from the glycolytic pathway and Krebs cycle in wildtype K. lactis, whereas glr1-deletion prevents this effect and actually causes up-regulation of the glycolytic, Krebs cycle, and oxidative pentose phosphate pathways. To our knowledge, this is the first global proteomic analysis performed on K. lactis. Keywords: oxidative stress • yeast • Kluyveromyces • proteomics • glutathione reductase
Introduction Studies of stress responses in human cell lines have led to apparently contradictory results owing to the complexity of their regulatory networks, making simpler model cell systems more useful.1 Yeasts are good models for studying the eukaryotic oxidative stress (OS) response since they offer defense mechanisms similar to those of higher eukaryotes and the tools for genome-wide experimental approaches are easily available. Many studies have been carried out on Saccharomyces cerevisiae, which has a predominantly fermentative metabolism, but information about these complex regulatory networks in other yeasts is more limited.2 Kluyveromyces lactis is a good alternative yeast model for analyzing the OS response, since it has a predominantly respiratory metabolism.3 Indeed, respiratory yeasts have been proposed as reliable models of neuronal cells, which also have a highly oxidative metabolism.4 Also, the complete genome of K. lactis has been sequenced5 and tools for genetic manipulation of this yeast are well developed.6 Reactive oxygen species (ROS) are byproducts of aerobic metabolism that modify various macromolecules chemically and cause cellular damage. Several human diseases have been related to ROS overproduction. An excess of ROS results in OS and may eventually cause cell death. Cells have evolved a number of mechanisms to counteract oxidative damage including the induction of antioxidant and redox enzymes and * To whom correspondence should be addressed. M.I.G.-S.,
[email protected]; A.G.-L.,
[email protected]; M.E.C.,
[email protected].
2358 Journal of Proteome Research 2010, 9, 2358–2376 Published on Web 03/29/2010
the maintenance of high levels of molecular scavengers such as glutathione (GSH). Oxidized thiol groups form mixed disulfides with GSH, which are then reduced by glutaredoxins and GSH reductase. ROS have toxic effects but also regulatory functions. Oxidation and reduction of protein thiol groups are thought to be the major mechanism by which ROS participate in cellular signal transduction pathways. Thiol groups are reported to have numerous roles within the cell and their redox status affects the activity and structure of many proteins including transcription factors and enzymes. In fact, most organisms have complex regulatory machineries, including GSH reductase, that maintain the correct redox status of SH groups in proteins and also lowmolecular-mass thiols.1,2 The sources of intracellular ROS are either exogenous or endogenous. Leakage of electrons from the mitochondrial respiratory chain has been described as the major source of endogenous ROS under physiological conditions.7 At a transcriptome level, OS caused by external agents induces a response in S. cerevisiae that affects many genes, with the participation of the general stress response factors Msn2 and Msn4 and the oxidative-stress-specific transcription factors Yap1 and Skn7.8-10 Recently, the transcription factors Mga2 and Rox1, previously shown to control the response to hypoxic conditions, have been shown to be active exclusively in the adaptive, not in the acute, response to OS, Mga2 being essential for adaptation.11 At a proteome level, exposure of yeast cells to H2O2 results in increased levels of antioxidant proteins, heat 10.1021/pr901086w
2010 American Chemical Society
research articles
Oxidative Stress Response in Kluyveromyces lactis shock proteins, components of the protein degradation machinery, and enzymes of the pentose phosphate pathway (PPP), which provides the NADPH necessary for some antioxidant enzymatic systems. Also, glycolysis and the protein translation apparatus are down-regulated.12-18 This proteome signature, although limited to about 20% of all proteins expressed in yeast cells, is approximately parallel to the previously described transcriptome pattern.10 Despite the above transcriptome/ proteome coincidences, recent studies based on the comparison of transcriptomes and proteomes from identical anaerobic and aerobic cultivation conditions yielded strong evidence for post-transcriptional regulation of key cellular processes such as glycolysis, aminoacyl-tRNA synthesis, purine nucleotide synthesis and amino acid biosynthesis.19 GSH reductase is a component of the so-called GSH/ glutaredoxin system. Glutaredoxins are small heat-stable thiol oxidoreductases that use the tripeptide GSH (gamma-glutamylcysteinyl-lysine) as a hydrogen donor. Reduced GSH is regenerated from glutathione disulfide (GSSG) by GSH reductase, which uses NADPH as a reducing source and FAD as a coenzyme. KlGLR1 (KLLA0E24069g) is the only gene from this system that has so far been experimentally studied in K. lactis.20 In K. lactis, KlGLR1 does not respond to peroxide treatment by changes in mRNA levels, nor is KlGlr1 enzyme activity modulated,21-23 whereas in S. cerevisiae Glr1 is an OS defenseinducible enzyme and GLR1 is a Yap1p target.24 Despite the lack of induction of KlGLR1 by peroxides, the influence of KlGlr1 on the OS response is inferred from the decrease in resistance to H2O2 in K. lactis when KlGLR1 is deleted and with the increase when KlGLR1 is overexpressed.23,25 In this work, we have studied the H2O2 regulon of the respiratory yeast K. lactis and the influence of deletion of the GSH reductase gene in this regulon by a proteomic approach. To our knowledge, this is the first proteome analysis and the first global analysis of the OS response in K. lactis.
Experimental Section Strains, Culture Conditions and Protein Extract Preparation. K. lactis wild-type (NRRL-Y1140, CBS 2359) and isogenic ∆glr1 mutant strains were used in this work. The construction of the ∆glr1 mutant is described in Garcı´a-Leiro et al.25 The cells were cultured in 500 mL flasks with 100 mL of synthetic complete medium (CM), prepared as described in Zitomer and Hall (1976),26 at 30 °C and 250 rpm. When OD600 reached 0.8, H2O2 was added to the cultures to a final concentration of 0.8 mM for one hour. The wild-type strain without H2O2 was used as control. Cellular protein extracts were prepared as described previously,21 and the proteins were precipitated with TCA and centrifuged at 9000× g. The pellet was solubilized by 1-h incubation with gentle agitation in an isoelectric focusingcompatible urea lysis buffer.27 To quantify protein, triplicate 2-4 µL samples of each extract were diluted to 50 µL with water and the BCA technique (Pierce) was used. Correct quantification was confirmed by loading 5 µg of each sample on a standard SDS-PAGE gel and subsequent Coomassie staining. DIGE Experimental Design and Protein Labeling. The proteomics comparison between (i) wild-type strain untreated with H2O2 (C), (ii) wild-type strain treated with H2O2 (C′) and (iii) ∆glr1 mutant treated with H2O2 (M′) was performed across six DIGE gels using the same pooled sample internal standard to reduce intergel variation. The 12 individual samples were generated from four independent cultures of each strain/ condition (C1 to C4, C′1 to C′4 and M′1 to M′4). Proteins in
each sample were tagged with a set of matched fluorescent dyes according to the manufacturer’s protocol for minimal labeling. To eliminate any dye-specific labeling artifacts, two samples of each group (C1 and C4, C′2 and C′4, M′1 and M′2) were labeled with Cy3 and the other two (C2 and C3, C′1 and C′3, M′3 and M′4) were labeled with Cy5. The pooled sample internal standard was always Cy2-labeled. In every case, 400 pmol of dye was used per 50 µg of protein. Briefly, labeling was performed for 30 min on ice in the dark, and the reaction was quenched with 1 µL 10 mM L-lysine for 10 min under the same conditions. 2-DE and Imaging of Cy-Labeled Proteins, DIGE Data Analysis, Mass Spectrometry and Database Search. These were performed by the procedures described in detail in RuizRomero et al. (2009).28 The samples were analyzed by MALDITOF MS. The searches for peptide mass fingerprints and tandem MS spectra were performed in the databases SwissProt release 57.4 (470369 sequences), MSDB release 20060831 (3239079 sequences) and NCBInr release 20090704 (9251875 sequences), with taxonomy restricted to fungi (23847, 175616, and 609216 sequences, respectively). Proteins identified by mass spectrometry, biological association network analysis and database search were mapped on to existing pathways and cellular networks through links at Ge´nolevures (http://www. genolevures.org/) and Saccharomyces Genome Database (SGD, http://www.yeastgenome.org/).
Results Effect of H2O2 on K. lactis Growth in Wild-Type and ∆glr1 Strains. We previously proved that 0.4 mM H2O2 induced the transcription of a group of genes related to the OS response in wild-type K. lactis21,22 as well as thioredoxin reductase and catalase activities23 and that 0.8 mM H2O2 slightly reduced the long-term growth of wild-type and ∆glr1 K. lactis strains on solid media.23,25 On the basis of these previous results, and to select the conditions for creating OS suitable for this K. lactis proteome analysis, we tested the effect of 0.8 mM H2O2 on short-term cell growth and viability. We measured OD600 and Colony-Forming Units during growth in liquid cultures at 1-h intervals in the wild-type and ∆glr1 strains. Growing cells were challenged with this concentration of H2O2 at OD600 ≈ 0.8. Supporting Information 1 shows a small inhibitory effect after 60 min; however, the differences were not statistically significant. Therefore, we selected as OS generating treatment by exposing cells, grown to an OD600 of approximately 0.8, to 0.8 mM H2O2 for 1 h. 2-D DIGE Analysis. Cells obtained from four individual CM cultures of both the K. lactis wild-type (C′1 to C′4) and ∆glr1 (M′1 to M′4) strains treated with H2O2, and cells from four CM cultures of the wild-type strain without H2O2 (C1 to C4), were used for protein isolation and DIGE-MS analysis. Supporting Information 2 describes the experimental procedure followed. After six-plex 2-D DIGE, three individual images were obtained from each gel, corresponding to Cy2-, Cy3- and Cy5-labeled samples. The 18 gel images were analyzed using DeCyder software. The differential in-gel analysis (DIA) module allowed an average of 2039 ( 131 (SD) protein spots to be detected on each image. Interimage spot matching was then carried out using the biological variation analysis (BVA) module. In this step, an average of 1535 ( 317 (SD) spots was matched on the gels, and their average abundances among the 18 images from our study were calculated. Statistical significance was assessed Journal of Proteome Research • Vol. 9, No. 5, 2010 2359
research articles
Garcı´a-Leiro et al.
Figure 1. Representative Cy2-labeled internal standard proteome map indicating the proteins altered in the K. lactis wild-type strain after treatment with H2O2. Proteins were resolved in the 3-11 (nonlinear) pH range in the first dimension and on 12% acrylamide gels in the second dimension. Proteins that exhibited a significant alteration in expression after treatment with H2O2 were identified by MALDI-TOF mass spectrometry and are listed in Table 1, where they are indicated by the same number as that in the figure.
for each change in spot abundance using Student’s t test and analysis of variance. We considered changes to be statistically significant and reproducible when they were within a 95% confidence interval (p < 0.05) and their standardized average spot volume ratios exceeded (1.3 in at least 80% of the gels analyzed (i.e., 12 of the 18 analyzed images). When proteins from the wild-type strain with and without H2O2 treatment were compared (C′/C), 57 spots increased and 52 decreased according to this analysis. These 109 spots are depicted on a DIGE gel image in Figure 1; Table 1 shows those that were identified by MALDI-PMF-MS. When proteins from the wild-type and ∆glr1 strains treated with H2O2 were compared (M′/C′), 64 spots were increased and 26 decreased in the mutant. These 90 spots are depicted on a DIGE gel image in Figure 2 and identifications are listed in Table 2. Multivariate Statistical Analysis. We sought to establish the biological significance of the protein changes by performing multivariate statistical tests on the proteins identified by DIGEMS. As shown in Figure 3, unsupervised principal component analysis (PCA) indicated how well the C and C′ groups were separated, that is, proteins of the wild-type strain without and with H2O2-treatment. Pattern analysis by hierarchical clustering (HC) (Figure 4) revealed two different patterns in this group of proteins and clearly differentiated two diverse profiles, clustering C and C′ as marked on the figure. The protein spot numbering in the heat map corresponds to the information in Table 1. As shown in Figure 5, PCA indicated how well the C′ and M′ groups were separated, that is, proteins of the wild-type and ∆glr1 mutant strains after H2O2-treatment. Pattern analysis by HC (Figure 6) revealed two different patterns in this group of proteins and clearly differentiated them, clustering C′ and M′ 2360
Journal of Proteome Research • Vol. 9, No. 5, 2010
as marked on the figure. The protein spot numbering in the heat map corresponds to the information in Table 2. Identification of Differentially Expressed Proteins. To identify the proteins, spots were selected, digested in-gel, and analyzed by MALDI-PMF-MS. A total of 33 of the 57 spots that increased upon H2O2 treatment (C′/C) were picked; very small spots were discarded. A Mascot database search using PMF (peptide mass fingerprinting) spectra allowed the proteins in 29 of the 33 spots obtained from duplicate gels to be identified. Table 1 shows detailed information comprising experimental and theoretical molecular weight and pI values, accession numbers and identification parameters. Four proteins could not be identified (spots 507, 1234, 1920 and 2036). In three cases (1205, 1226 and 1698), two different proteins were identified on the same spot. We also found four redundancies (i.e., the same protein identified in several spots), that might be attributable to posttranslational modifications or proteolysis. Finally, 12 different protein isoforms were assigned by estimation of experimental molecular weights and pI differences on the gels. A total of 26 of the 52 spots that decreased upon H2O2 treatment (C′/C) were picked; very small spots were discarded. Ten proteins were identified from M′/C′ gels as indicated in Table 1. A Mascot database search using the PMF spectra allowed the proteins present in the 26 spots obtained from duplicate C′/C gels to be identified (Table 1). In two cases (spots 784 and 1346), two different proteins were identified on the same spot, and we also found six redundancies. Also, 12 different protein isoforms were identified as listed in Table 1. A total of 35 of the 64 spots that were increased in the H2O2treated ∆glr1 mutant vs the wild-type strain (M′/C′) were picked; very small spots were discarded. Five proteins also increased upon H2O2 treatment (C′/C) and are identified in
no.
568
649
907
908 1180 1198
1199
1205
1205 1222
1225
1226
1226
1231
1237
1238
1256
1291
1440
1465 1476
1488
1494 1503 1506 1509
1518
1698
1698 1701
pos.
12
17
31
32 43 49
50
51
51 52
53
54
54
56
58
59
60
61
68
69 71
73
74 75 76 77
78
82
82 83
18 18
18
18
18 18 18 18
18
18 18
18
18
18
18
18
18
18
18
18
18 18
18
18
18 15 18
18
18
18
app.
1.69
2.09
2.09 1.93
3.00 × 10-5
3.00 × 10-5 0.00066
12.82 7.85 4.3 1.36
18.55
2.91 1.35
0.0013
5.40 × 10 2.20 × 10-5 1.20 × 10-5 0.039
-6
2.10 × 10-6
0.0011 0.017
1.53
1.31
6.30 × 10-5
0.00016
1.8
1.73
1.75
2.27
2
2
2.22
2.25 1.56
2.25
0.0006
0.0014
0.00034
0.00031
4.70 × 10-7
4.70 × 10
-7
5.00 × 10 0.0029
0.00061
-5
5.00 × 10
3.37
1.00 × 10-5
-5
1.57 2.86 2.67
1.36
0.0047 3.70 × 10-5 1.70 × 10-5
0.0006
1.59
1.42
3.20 × 10-5
0.00038
av.
t test
-7
1.40 × 10-7 0.00014
1.40 × 10-7
0.0041
2.10 × 10 1.40 × 10-6 1.10 × 10-6 0.0012
-7
5.50 × 10-8
2.50 × 10 0.021
-5
2.30 × 10-8
0.00032
1.20 × 10-5
0.0043
8.20 × 10-6
3.20 × 10-5
2.70 × 10-8
2.70 × 10
-8
2.50 × 10-5
4.10 × 10 0.00011
4.10 × 10
-7
5.50 × 10-8
0.00043 1.20 × 10-5 2.00 × 10-7
0.0048
0.0036
1.10 × 10-5
1-ANOVA
YDL124w, NADPH-dependent alpha-keto amide reductase )1488, YDL124w )1488, YDL124w )1488, YDL124w SNZ3, pyridoxal 5′-phosphate (PLP) synthase subunit SNZ3, pyridoxal 5′-phosphate (PLP) synthase subunit RPL2B, 60S large subunit ribosomal protein RPS4A, 40S ribosomal protein S4 )1698, RPL2B/RPS4A
UGA1, 4-aminobutyrate aminotransferase )907, UGA1 PGK1, 3-phosphoglycerate kinase ADH6, NADPH-dependent medium chain alcohol dehydrogenase YNL134c, putative alcohol dehydrogenase (NADP+) YNL134c, putative alcohol dehydrogenase (NADP+) PGK1, 3-phosphoglycerate kinase SER1, phosphoserine transaminase KYE1, NADPH dehydrogenase (old yellow enzyme) GRE2, NADPH-dependent oxidoreductase HOM2, aspartate-semialdehyde dehydrogenase KYE1, NADPH dehydrogenase (old yellow enzyme) GRE2, NADPH-dependent oxidoreductase KYE1, NADPH dehydrogenase (old yellow enzyme) YNL134c, putative alcohol dehydrogenase (NADP+) ILV5, acetohydroxi-acid reducto-isomerase ZTA1, NADPH:quinone reductase, zeta Crystallin homologue )1476, TRR1 TRR1, thioredoxin reductase
SSA3, heat shock protein of HSP70 family LEU4, 2-isopropylmalate synthase
function or homology
KLLA0B03652g
KLLA0D16027g
KLLA0A00374g
KLLA0A00374g
KLLA0F05775g
KLLA0E21692g
KLLA0A10549g
KLLA0A02673g
KLLA0F24464g
KLLA0A09075g
KLLA0D11000g
KLLA0A09075g
KLLA0D16731g
KLLA0D11000g
KLLA0A09075g
KLLA0A11011g KLLA0C04818g
KLLA0F24464g
KLLA0F24464g
KLLA0A11011g KLLA0E19987g
KLLA0F20548g
KLLA0D14201g
KLLA0E20603g
Locus-tag ge´nolevures
translation
translation
vitamin B6 metabolism
vitamin B6 metabolism
cellular redox homeostasis/ response to oxidative stress cellular metabolic process/ response to oxidative stress
valine, leucine and isoleucine biosynthesis oxidation reduction (response to oxidative stress)
oxidation reduction
celular metabolic process (response to stress) oxidation reduction
cellular metabolic process (response to stress) glycine, serine and threonine metabolism oxidation reduction
glycolysis/gluconeogenesis glycine, serine and threonine metabolism oxidation reduction
oxidation reduction
oxidation reduction
glycolysis/gluconeogenesis oxidation reduction
ATP binding (Protein folding and response to stress) valine, leucine and isoleucine biosynthesis alanine, aspartate and glutamate metabolism
KEGG-pathway or biological process (Uni-Prot)
Part A: Proteins that increase their expression
89
144
135
152
345
147
110
291
219
166
164
244
72
75
323
101 245
164
269
152 199
188
75
250
sc.
11/50
13/50
15/36
20/50
25/50
13/50
10/34
26/50
16/47
14/39
16/50
22/50
7/32
8/32
27/50
11/50 21/50
15/50
21/50
14/50 21/50
18/47
8/26
22/50
pep.
Table 1. K. lactis Proteins Identified by 2-D DIGE-MS as Differentially Expressed upon H2O2 Treatment and Their Predicted Biological Functiona
44
55
38
48
87
53
31
61
58
41
65
56
28
32
63
44 56
56
72
50 63
37
20
46
cov.
26.8 26.8
26.8
33.8
32.4 32.5 32.5 33.8
32.4
34.6 34.2
35.5
36.8
36.9
37.2
37.0
37.2
37.0
37.0
37.2
37.2 37.1
37.2
37.2
50.5 37.4 37.2
50.5
60.3
71.6
Mr (E)
5.04 5.01
5.04
5.00
5.74 5.72 5.52 5.02
5.92
5.23 5.20
5.74
5.16
5.96
5.21
5.74
5.27
5.62
5.62
5.37
5.86 5.88
5.86
5.96
5.43 5.96 5.83
5.54
5.22
4.91
pI (E)
29.6
27.4
32.2.
32.2
35.0
38.3
38.7
44.3
40.1
44.8
40.2
44.8
39.8
40.2
44.8
44.5 43.2
40.1
40.1
44.5 40.9
52.9
65.6
70.2
Mr (P)
10.18
11.10
5.23
5.23
6.06
5.74
7.68
7.68
6.14
5.60
5.91
5.60
5.74
5.91
5.60
6.63 6.12
6.14
6.14
6.63 5.96
5.66
5.30
4.99
pI (P)
Oxidative Stress Response in Kluyveromyces lactis
research articles
Journal of Proteome Research • Vol. 9, No. 5, 2010 2361
2362
no.
1707 1871
1877 1889 1894
1896 1897 1929
1937 1944
1991
2005
2206
2208
no.
306 308 380 406 629
680 709 723 726 734 741 784
784 798 827 830 837 838 852
1040 1077
1111 1152
pos.
84 91
92 94 95
96 97 100
101 102
103
104
Journal of Proteome Research • Vol. 9, No. 5, 2010
107
108
pos.
4 5 7 8 15
18 19 20 21 22 23 24
24 25 26 27 28 29 30
33 34
36 37
18 18
18 18
18 15 18 18 18 18 18
18 18 18 18 18 18 18
18 18 18 18 18
app.
18
18
18
18
18 18
18 18 18
18 15 18
18 18
app.
0.017 0.0073
0.0024 0.00091
0.016 0.037 0.0097 0.0053 0.01 0.0013 0.002
0.0036 0.0041 0.0048 0.0057 0.0022 0.01 0.016
0.00093 0.0042 0.0068 0.0046 0.0023
t test
3.80 × 10
1-ANOVA
0.045 0.019 0.0038 0.0054 0.035
0.0028 0.00057 0.0027 0.025 0.0017 0.0061 0.044
0.044 0.028 0.0081 0.0031 0.021 0.00069 0.0059
0.0052 0.00049
0.0086 0.0029
-1.32 -1.32 -1.31 -1.32 -1.33
-1.32 -1.46 -1.6 -1.4 -1.67 -1.42 -1.41
-1.41 -1.44 -1.73 -1.83 -1.34 -1.78 -1.54
-1.42 -1.49
-1.6 -1.34
5.50 × 10
-7
3.30 × 10-7
0.0012
0.0009
6.20 × 10-7 2.10 × 10-6
KLLA0F06732g (91% similarity)
KLLA0F06732g
KLLA0F06732g
KLLA0F20009g
KLLA0F04323g
KLLA0C01562g
KLLA0B01628g
KLLA0A00352g
Locus-tag ge´nolevures
negative regulation of transcription cell redox homeostasis/response to oxidative stress glutathione metabolism/response to oxidative stress glutathione metabolism/response to oxidative stress glutathione metabolism/response to oxidative stress
unknown
cell redox homeostasis/response to oxidative stress
vitamin B6 metabolism
KEGG-pathway or biological process (Uni-Prot)
PYK1, pyruvate kinase )706b, PDC1 )709, PDC1 PYK1, pyruvate kinase )734b, PYK1 PYK1, pyruvate kinase ALD5, mitochondrial aldehyde dehydrogenase SES1, seryl-tRNA synthetase )784, SES1 )837, TRR1/HXK2 )837, TRR1/HXK2 )837b, TRR1/HXK2 )837, TRR1/HXK2 ALD5, mitochondrial aldehyde dehydrogenase )1040b, ENO1 ERG10, acetyl-CoA C-acetyltransferase )1153, PGK1 PGK1, 3-phosphoglycerate kinase
)308, EFT1 )308b, EFT1 )380b, MET6 )406b, ACO1 ILV3, dihydroxy-acid dehydratase
function or homology
glycolysis/gluconeogenesis glycolysis/gluconeogenesis oxidation reduction
KLLA0F23397g KLLA0F23397g KLLA0C07777g
KLLA0A11011g
glycolysis/gluconeogenesis
fatty acid metabolism
oxidation reduction
KLLA0C07777g KLLA0D12056g
translation
KLLA0A10593g
KLLA0F23397g
valine, leucine and isoleucine biosynthesis glycolysis/gluconeogenesis
KEGG-pathway or biological process (Uni-Prot)b
KLLA0F22022g
Locus-tag ge´nolevures
Part B: Proteins that decrease their expression
GPX3, glutathione peroxidase
HYR1, glutathione peroxidase
AHP1 (PRX5), alkyl hydroperoxide reductase (peroxiredoxin) HYR1, glutathione peroxidase
)1894, TSA1 )1894, TSA1 YCL026cb Pi, nitroreductase-like family 4 )1929, YCL026cb Pi PST2, flavodoxin
4.80 × 10-5 4.00 × 10-8 9.50 × 10-9
3.20 × 10-8 7.80 × 10-6 4.30 × 10-8
)1698, RPL2B/RPS4A SNO3, glutamine amidotransferase )1871, SNO3 )1894, TSA1 TSA1, peroxiredoxin
function or homology
Part A: Proteins that increase their expression
3.90 × 10-7 1.10 × 10-7
1-ANOVA
av.
6.12
5.68
4.80 × 10-7
-7
4.6
1.62
0.00069
0.0011
4.01 3.13
2.70 × 10-5 0.0003
3.17 2.73 2.89
4.40 × 10-6 0.00023 6.10 × 10-6
1.74 2.63 4.14
2.04 3.7
1.80 × 10-5 1.50 × 10-5
0.0022 6.80 × 10-6 3.80 × 10-6
av.
t test
Table 1. Continued
344
241
269
87
353 98
369
121
267
sc.
70
192
59
104
131
146
127
129
sc.
24/50
18/50
23/50
11/50
22/50 12/50
31/50
11/49
25/45
pep.
6/50
13/50
5/30
7/50
10/50
10/36
10/50
11/50
pep.
75
74
62
30
68 32
67
36
50
cov.
33
59
27
84
79
62
50
87
cov.
38.9 38.4
46.5 42.0
52.7 52.7 52.8 52.8 52.8 52.8 51.8
57.1 58.1 57.9 55.3 55.2 55.1 52.7
93.7 93.8 86.5 86.2 58.8
Mr (E)
18.0
18.0
17.8
18.5
19.9 19.1
21.6 22.5 19.9
21.9 21.7 21.6
26.8 22.6
Mr (E)
7.03 8.31
5.30 7.02
5.68 5.60 5.09 5.07 5.05 5.01 5.59
5.61 5.11 5.13 5.74 5.61 5.50 5.68
5.95 6.03 5.74 6.87 7.27
pI (E)
5.18
5.34
5.62
4.90
5.13 5.58
4.89 4.93 5.27
5.18 4.99 4.96
4.99 5.34
pI (E)
44.5
41.3
56.8
53.2
55.2 56.8
55.2
55.2
63.0
Mr (P)
18.8
18.5
18.5
18.5
21.2
21.1
21.6
24.4
Mr (P)
6.63
6.61
6.06
5.65
5.85 6.06
5.85
5.85
7.17
pI (P)
8.30
5.63
5.63
5.12
5.60
5.56
4.96
5.21
pI (P)
research articles Garcı´a-Leiro et al.
1153 1159 1170 1176 1177 1185 1193 1194 1195 1196 1325 1342 1345 1346 1346 1596 1620 1622 1782 1796 1832 1838 1840 1847 1879 2210
38 39 40 41 42 44 45 46 47 48 63 64 65 66 66 79 80 81 85 86 87 88 89 90 93 109
18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 15
app.
0.0017 0.002 1.70 × 10-5 0.00026 0.00087 0.0032 0.0025 0.00085 4.20 × 10-5 0.00015 0.011 0.0024 0.03 0.028 0.028 0.028 0.038 0.013 0.041 0.00088 0.046 0.0057 0.0047 0.0021 0.0092 0.006
t test
1-ANOVA
0.0012 0.00022 6.40 × 10-6 4.70 × 10-5 9.10 × 10-5 0.00026 0.0026 0.0023 7.50 × 10-5 0.00011 0.012 0.0097 0.00049 0.002 0.002 5.20 × 10-5 0.00026 0.034 0.11 1.80 × 10-5 0.016 0.0029 6.00 × 10-5 0.0021 0.00019 0.018
av.
-1.49 -1.33 -1.97 -1.52 -1.52 -1.42 -1.96 -1.81 -1.78 -1.96 -1.42 -1.52 -1.32 -1.37 -1.37 -1.48 -1.6 -1.34 -1.3 -1.41 -1.39 -1.47 -1.33 -1.35 -1.31 -1.35 PGK1, 3-phosphoglycerate kinase )1153, PGK1 ADH1, Alcohol dehydrogenase I )1170, ADH1 ADH1, Alcohol dehydrogenase I ADH1, Alcohol dehydrogenase I )1193b, ADH2 )1193, ADH2 )1193, ADH2 )1193, ADH2 FBA1, Fructose-1,6-bisphosphate aldolase FBA1, Fructose-1,6-bisphosphate aldolase )1346, RPL4B/ADO1 RPL4B, 60S large subunit ribosomal protein ADO1, Adenosine kinase )1598b, RHR2 )1622b, RHR2 SPE3, spermidine synthase SEC53, phosphomannomutase ADK1, adenylate kinase TPI1, Triosephosphate isomerase TPI1, Triosephosphate isomerase TPI1, Triosephosphate isomerase TPI1, Triosephosphate isomerase FUR1, uracil phosphoribosyltransferase COF1, cofilin
function or homology
230 18/50 168 14/50
cysteine and methionine metabolism fructose and mannose metabolism purine metabolism glycolysis/gluconeogenesis glycolysis/gluconeogenesis glycolysis/gluconeogenesis glycolysis/gluconeogenesis pyrimidine salvage cell cycle (actin binding)
21/48 17/50 20/43 16/50 19/50 21/50 15/50 22/50 9/50
250 205 211 199 281 336 185 271 137
glycolysis/gluconeogenesis glycolysis/gluconeogenesis
KLLA0E07546g KLLA0E07546g
292 23/50 223 18/50
303 23/50
KLLA0B09372g KLLA0D05709g KLLA0F13376g KLLA0F18832g KLLA0F18832g KLLA0F18832g KLLA0F18832g KLLA0E22990g KLLA0E00396g
glycolysis/gluconeogenesis glycolysis/gluconeogenesis
KLLA0F21010g KLLA0F21010g
pep.
160 16/50 153 16/50
glycolysis/gluconeogenesis
KLLA0F21010g
sc.
313 24/50
KLLA0B07139g translation KLLA0D10890g purine metabolism
glycolysis/gluconeogenesis
pathway or biological processb
KLLA0A11011g
Locus-tag ge´nolevures
Part B: Proteins that decrease their expression
65 73 66 49 64 78 46 89 56
52 47
70 60
62 62
62
72
37.6 37.6 37.6 37.5 37.6 37.5 37.5 37.5 37.5 37.5 36.3 36.1 36.0 36.0 36.0 30.5 30.2 30.0 25.2 23.5 23.2 23.1 23.1 23.1 21.6 36.1
7.03 6.99 5.76 5.68 5.61 5.47 5.28 5.26 5.23 5.18 6.15 5.93 4.30 4.32 4.32 5.21 5.32 5.08 5.98 6.21 5.70 5.50 5.32 5.20 5.69 5.49
33.4 29.0 25.5 27.1 27.1 27.1 27.1 24.8 16.1
39.2 46.9
39.7 39.7
37.6 37.6
37.6
44.5
5.24 5.11 6.53 5.50 5.50 5.50 5.50 5.96 4.96
10.71 6.14
5.91 5.91
5.77 5.77
5.77
6.63
cov. Mr (E) pI (E) Mr (P) pI (P)
a No., master protein spot number; pos., protein spot number according to Figures 2 and 5; app, number of gel images (from a total of 18) where each spot appears; av., average volume ratio C′/C quantified by DeCyder BVA module; sc., Mascot MS protein score. In all cases, a probability score
