Proteomic and Functional Characterization of a - American Chemical

Apr 21, 2010 - Plant Biochemistry, Laboratory of Mass Spectrometry, and GIGA-Neuroscience, University of Liege, Belgium. Received September 25, 2009...
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Proteomic and Functional Characterization of a Chlamydomonas reinhardtii Mutant Lacking the Mitochondrial Alternative Oxidase 1 Gre´gory Mathy,† Pierre Cardol,‡ Monique Dinant,‡ Arnaud Blomme,† Ste´phanie Ge´rin,† Marie Cloes,† Bart Ghysels,§ Edwin DePauw,| Pierre Leprince,⊥ Claire Remacle,‡ Claudine Sluse-Goffart,† Fabrice Franck,§ Rene´ F. Matagne,*,‡ and Francis E. Sluse*,† Laboratory of Bioenergetics and Cellular Physiology, Laboratory of Genetics of Microorganisms, Laboratory of Plant Biochemistry, Laboratory of Mass Spectrometry, and GIGA-Neuroscience, University of Liege, Belgium Received September 25, 2009

In the present work, we have isolated by RNA interference and characterized at the functional and the proteomic levels a Chlamydomonas reinhardtii strain devoid of the mitochondrial alternative oxidase 1 (AOX1). The AOX1-deficient strain displays a remarkable doubling of the cell volume and biomass without alteration of the generation time or change in total respiratory rate, with a significantly higher ROS production. To identify the molecular adaptation underlying these observations, we have carried out a comparative study of both the mitochondrial and the cellular soluble proteomes. Our results indicate a strong up-regulation of the ROS scavenging systems and important quantitative modifications of proteins involved in the primary metabolism, namely an increase of enzymes involved in anabolic pathways and a concomitant general down-regulation of enzymes of the main catabolic pathways. Keywords: Mitochondria • Alternative oxidase (AOX) • Proteomics • Metabolism

Introduction The mitochondrial alternative oxidase (AOX) is a terminal ubiquinol oxygen oxidoreductase that bypasses complex III and complex IV of the respiratory chain. Its activity generates an electron partioning between the cytochrome pathway and AOX itself, which ultimately leads to a decrease of the proton pumping efficiency per electron of the respiratory chain. Thus, the activity of AOX is not coupled to ATP production and the free energy produced is dissipated as heat. This is responsible for thermogenesis in specialized tissue where AOX is very abundant (spadix of Araceae). Two important roles have been attributed to AOX in nonthermogenic tissues and cells. First, by maintaining an appropriate ratio between the reduced and oxidized forms of ubiquinone,1 the activation of AOX could be a mechanism preventing the over-reduction of respiratory chain components that might otherwise result in the production of harmful reactive oxygen species (ROS).2,3 Second, the mitochondrial ubiquinone pool can be considered as a redox power bottleneck of the energy metabolism, accepting electrons from several types of dehydrogenases. Consequently, the reoxidation of ubiquinol by AOX would also allow the regeneration of oxidized cofactors upstream of the respiratory chain in case of high reduced substrate supply and/or high phosphate potential, allowing continuous operation of glycolysis and tricarboxylic acid cycle.4 In higher plants, a wide range of stress * To whom correspondence should be addressed. E-mail: (F.E.S.) [email protected] and (R.F.M.) [email protected]. † Laboratory of Bioenergetics and Cellular Physiology, University of Liege. ‡ Laboratory of Genetics of Microorganisms, University of Liege. § Laboratory of Plant Biochemistry, University of Liege. | Laboratory of Mass Spectrometry, University of Liege. ⊥ GIGA-Neuroscience, University of Liege. 10.1021/pr900866e

 2010 American Chemical Society

factors are known to enhance Aox gene transcription and/or AOX protein expression. They include chilling,5 oxidative stress,2,6 saline stress,7 exposure to heavy metals,8 phosphate limitation, and inhibition or restriction of the cytochrome pathway,4,9,10 All these studies have reported thoroughly the different possible roles of AOX in the cell physiology, indicating that, in addition to the uncoupling protein, AOX constitutes the last control point of the aerobic metabolism balance and enables to maintain an optimal metabolic steady state in order to face several kinds of stresses. Thus, disruption of the oxidative phosphorylation yield via the overexpression or inactivation of energy dissipating systems may have a subtle influence on upstream metabolic pathways to counterbalance changes in the expression level of these systems. Moreover, since an important number of factors are likely to be affected in such situations, the utilization of global techniques of investigation, like comparative proteomics,11 appears to be a suitable choice to identify important metabolic pathways that could be affected. In this context, we have previously used 2D-DIGE to study the impact of the heterologous expression of AOX on the mitochondrial proteome of Saccharomyces cerevisiae, which is naturally devoid of this enzyme. Our proteomics survey has clearly highlighted that the ectopic expression of AOX in yeast leads to numerous up-regulations of proteins mostly involved in the primary metabolism.12 Among them, several enzymes of the Krebs cycle were found to be up-regulated to provide additional reduced cofactors to the respiratory chain in order to counterbalance the electron leak due to the activity of AOX. These observations are quite important since they show that organisms do not undergo the ectopic expression passively, but Journal of Proteome Research 2010, 9, 2825–2838 2825 Published on Web 04/21/2010

research articles actively adapt in order to counterbalance the activity of heterologous proteins.12,13 In the unicellular green alga Chlamydomonas reinhardtii, AOX is a monomeric protein that is potentially encoded by two genes, Aox1 and Aox2.14,15 Transcriptional analyses have shown that only the Aox1 gene is significantly expressed.14–16 Apparently peculiar to C. reinhardtii is the regulation by the nitrogen source of Aox1 gene transcription and AOX protein expression:15,17 AOX protein is much more abundant in cells grown in nitratecontaining medium compared to cells grown in ammoniumcontaining medium. Moreover, this unicellular organism offers the opportunity to use RNA interference to inactivate selectively the expression of a given gene. In the present study, we have isolated a strain of C. reinhardtii lacking AOX1 as a result of the silencing of the Aox1 gene. At the functional level, we have analyzed several parameters previously reported to be potentially affected by the lack of AOX1. The results obtained were in good agreement with data reported in the literature. We have also observed a remarkable enhancement of the cell size in association with a biomass increase. As a logical continuation of AOX ectopic expression in S. cerevisiae (creation of function), we have undertaken a comparative study of both the mitochondrial and the cellular proteomes to highlight the possible molecular reorganization set up by the cell to counterbalance the lack of AOX1 (loss of function). Our proteomics data clearly indicate a decrease of the most important catabolic pathways such as the Krebs cycle or acetate assimilation and a general upregulation of the anabolic pathways.

Material and Methods Chlamydomonas Strains and Culture Conditions. The host strain used in cotransformation experiments is the wall-less, arginine-requiring cw15 arg7-8 mt+ mutant, deficient for argininosuccinate lyase (strain 325). The wild-type wall-less cw15 mt+ (strain 83) was used as a control. Both strains can utilize nitrate or ammonium as a nitrogen source. Cells were routinely grown at 25 °C on solidified agar under 50 µmol m-2 s-1 continuous white light. Two media were used depending on the nitrogen source: the TAP NH4 medium containing 7.5 mM NH4Cl and the TAP NO3 medium containing 4 mM KNO3.18 Arginine (60 µM) was added to the medium when required. For liquid cultures, Erlenmeyer flasks were seeded with cells from agar plate cultures at a starting concentration of 4 × 105 cells mL-1 and cultures were carried out on a rotary shaker under 50 µmol m-2 s-1 continuous light. Cell densities and mean cell volumes were determined using a Coulter counter (Coulter Electronics, Harpenden Herts, U.K.). Turbidity (750 nm) was proportional to the cellular volume and the number of cell per milliliter. Sense-Antisense Construction. The Aox1 gene sequence comprises 8 exons and 7 introns.14 The EcoRI-BamHI genomic fragment (3189 bp) including the 0.9 kb promoter region, the four first exons, and the 5′ end of exon 5 of the Aox1 gene was inserted in the pBluescript KS(+) plasmid vector (Stratagene). At the BamHI site of this new plasmid, we introduced in antisense orientation a 679 bp cDNA fragment covering the exons 1-5 of Aox1. This new plasmid was called pULGD22. Cotransformation Experiments. The nuclear transformation of the 325 strain was performed following the glass bead method,19 using 1 µg of pASL plasmid20 bearing the wild-type Arg7 gene as a selectable marker and 3 µg of pULGD22 plasmid. After treatment, cells were spread onto TAP NH4 agar plates 2826

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Mathy et al. for selection of arginine-independent transformants. To test the sensitivity of transformants to antimycin A (1 µM) and myxothiazol (7.5 µM), inhibitors of complex III, drops of cell suspensions containing (1-1.5) × 107 cells/mL were plated on TAP NH4 agar added with the inhibitors and growth was estimated after 3-5 days. Protein, Chlorophyll, and Starch Content. Protein content was determined using the method described by Bradford and co-workers.21 Pigments were extracted from whole cells with ethanol. After centrifugation at 10 000g for 5 min, the chlorophyll (a+b) concentration in the supernatant was determined according to Lichtenthaler.22 For starch content determination, (2-5) × 106 cells were extracted with ethanol at 45 °C for 3 h, and after centrifugation, starch present in the pellet was determined using the phenol-sulfuric acid method as described in Matagne et al.23 Western-Blot Analysis. Western-blot analysis was performed as described previously by Baurain and co-workers.15 The polyclonal antibodies raised against AOX of C. reinhardtii were obtained from Dr. S. Merchant, University of California, Los Angeles, CA, and used at a dilution of 1:50 000. Isolation and Purification of Mitochondria. Crude mitochondria fractions were isolated from cell wall-less strains according to a published procedure24 and were used for oxygen consumption measurements. They were further purified for the proteomic analysis using the procedure of Eriksson and coworkers25 with some modifications: crude mitochondria were loaded on a Percoll gradient made of three layers (13%, 21%, and 45% Percoll in a 10 mM Tris buffer, pH 7.0, containing 280 mM mannitol, 0.1% bovine serum albumin, and 500 µM EDTA), and then centrifuged for 50 min at 40 000g. Mitochondria were collected at the interface of 21% and 45% Percoll layers, diluted with 40 mL of buffer without bovine serum albumin, and spun for 10 min at 10 000g. Respiration Measurements and Light-Saturation Curves. Cellular and mitochondrial respiratory rates were measured as oxygen uptake in the dark at 25 °C using a Clark-type oxygen electrode (Hansatech, King’s Lynn, U.K.). For measurements of whole-cell respiratory rates, cells in exponential growth phase were harvested by centrifugation and resuspended into fresh medium at a density of 5 × 106 cells mL-1. The cytochrome pathway and AOX were, respectively, inhibited by 1 mM KCN in aqueous solution and 1 mM salicylhydroxamic acid (SHAM) in DMSO (final concentration 0.5%). The possible inhibitory effect of 0.5% DMSO alone (ranging between 0 and 10% depending on the assays) was subtracted from the measurements. For measurements of state III mitochondrial respiratory rates, crude mitochondria extracted following the procedure described above were loaded in 1 mL of buffer containing 30 mM KCl, 4 mM EDTA, 10 mM MgCl2, 57.5 mM Tris, 2.5 mM KH2PO4, 5 µM rotenone, 10 mM succinate, and 5 mM NADH, pH 7.4. The cytochrome pathway and AOX were, respectively, inhibited by 1 mM KCN in aqueous solution and 2.3 mM benzohydroxamic acid (BHAM) in ethanol. State III respiration was induced by addition of 500 nmol ADP. For both the cellular and the mitochondrial assays, we measured the total respiration and the respiration after addition of cyanide first and then SHAM or BHAM, and vice versa. The cytochrome pathway respiration corresponds to the respiration after addition of SHAM or BHAM, which can be inhibited by cyanide, whereas AOX respiration is the respiration after addition of cyanide, which can be inhibited by SHAM or BHAM.

Proteomic/Functional Characterization of a C. reinhardtii Mutant Light-saturation curves of photosynthetic oxygen evolution were measured on whole-cells using a Clark-type electrode (Chlorolab 2, Hansatech) as described by Cardol et al.26 ROS Measurements. Peroxide and superoxide anion levels were determined by in vivo staining with 3-3′ diaminobenzidine (DAB) HCl (Sigma) and nitroblue tetrazolium (NBT), respectively.27 Equal amounts of biomass (equivalent to 1 mL of the culture leading to an absorbance of 2 at 750 nm) from log phase cell cultures were filtered onto GVWP durapore membrane filters (0.22 µm pore size, 47 mm Ø, Millipore). Filters were moistened with 400 µL of culture medium containing either 5 mM DAB or 1 mM NBT and incubated 1 h in the dark, 20-40 min in moderate light (50 µmol m-2 s-1), or 5-20 min in high light (200 µmol m-2 s-1). Staining reactions were then stopped by gently washing the filters with methanol, and stained filters were subjected to densitometry analysis with Genesnap/ Genetool softwares from Syngene. Samples Preparation for the Comparative Proteomics Studies. Freshly harvested cells or purified mitochondria were resuspended in a lysis buffer (7 M urea, 2 M thiourea, 2% ASB14, 10 mM dithiothreitol (DTT), antiprotease cocktail Complete EDTA Free (Roche), 0.5 mM EDTA, 50 mM TRIS, pH 7.5) and intensively vortexed for 30 min at room temperature. The suspensions were briefly sonicated and centrifuged at 10 000g to remove any insoluble material.13 To remove excess of salts, fatty acids, chlorophylls and nucleic acids, protein extracts were precipitated and cleaned using the 2D Clean-Up Kit (GE Healthcare). Protein pellets were resuspended in the 2D-DIGE label buffer (7 M urea, 2 M thiourea, 2% ASB-14, Complete EDTA Free (Roche), 0.5 mM EDTA, 50 mM TRIS, pH 8.5). Exact protein concentration was measured using RC/DC Protein Assay Kit (BioRad Laboratories) and was adjusted between 5 and 10 mg/mL. 2D-DIGE Electrophoresis. For the 2D-DIGE comparative proteomic experiments on both the mitochondrial and the cellular proteomes, a set of 3 independent cultures in each experimental condition (wild-type and AOX1-) has been carried out. For analytical gels, 25 µg of each biological replicate was either labeled with 0.2 nmol of Cy3 or Cy5 (minimal labeling) for 30 min at room temperature in the dark. To avoid any fluorescence artifact, an inversion of labeling was carried out: for each biological replicate from both strains, 25 µg was labeled with Cy3 and 25 µg with Cy5. At the same time, an internal standard (used for matching and normalization between gels) constituted of an equimolar amount of all the biological replicates from wild-type and AOX1- strains was labeled with Cy2. For preparative gels, 750 µg of proteins constituted of an equimolar amount of all the biological replicates was used, out of which 25 µg was labeled with 0.2 nmol of Cy2 (minimal labeling). Labeling reactions were stopped by adding 5 nmol of lysine. For analytical gels, Cy2, Cy3 and Cy5 labeled proteins were pooled together before the isoelectrofocusing (IEF). Samples were reduced by adding 10 mM DTT and resuspended in rehydration buffer (7 M urea, 2 M thiourea, 2% ASB-14, and 0.6% IPG Buffer pH 3-7 NL or pH 3-11 NL (GE Healthcare)) to reach a final volume of 450 µL, and laid on a 24 cm regular strip holder (GE Healthcare). The 3-7 NL or 3-11 NL IPG Drystrips (GE Healthcare) were passively rehydrated in the strip holder for 10 h before IEF. IEFs were run in the following condition at 20 °C: 200 V for 200 Vh (step), 500 V for 500 Vh (Gradient), 500 V for 500 Vh (Step), 1000 V for 2000 Vh (Gradient), 8000 V for 8000 Vh (gradient) and 8000 V for 60 000 Vh (Step) with a maximum current setting fixed at 50 µA.28

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After the first dimension, strips were equilibrated before the second dimension. Strips were reduced in an equilibration buffer (50% glycerol (v/v), 2% SDS (w/v), 6 M urea, 50 mM TrisHCl, pH 8.8) containing 300 mM DTT (Sigma) for 20 min. Then, strips were alkylated in the same buffer containing 350 mM iodoacetamide (Sigma) for 20 min. After equilibration, strips were laid on the top of a 12.5% (w/v) acrylamide gel in Laemmli SDS electrophoresis buffer (25 mM Tris, 192 mM glycine, 1% SDS (w/v)). For preparative gels, one plate was treated with bind silane for spot picking. Electrophoresis was carried out overnight at 1.5 W/gel (constant power). Protein Detection and Quantification. Gels were scanned with a Typhoon 9400 scanner (GE Healthcare) at the specific excitation wavelength of each CyDye. Since an inversion of labeling was carried out, 18 gel images were obtained for each proteome (2 × 3 images for the wild-type strain, 2 × 3 images for the AOX1- strain and 6 images for the internal standard). Images were analyzed with the DeCyder V6.5 software (GE Healthcare). In brief, codetection of the three CyDye-labeled forms of each spot was performed using the DIA (Differential In-gel Analysis) module. The DIA module performs the spot detection, the ratio calculation, and the spot abundance normalization via the internal standard. Statistical analysis was carried out in the BVA (Biological Variation Analysis) module after intergel matching. Protein spots that showed a statistically significant Student’s t-test (p < 0.05, n ) 6) for an increase or decrease ranging up to +1.2 or down to -1.2 in normalized ratio intensity were accepted as being differentially expressed between wild-type and AOX1- strains. In-Gel Digest and Mass Spectrometry. Matched spots presenting a statistical difference between wild-type and AOX1experimental groups were picked using the Ettan Dalt Spot Picker (GE Healthcare). Proteins in gel pieces were subsequently in-gel digested according to Shevchenko and colleagues,29 with some modifications. Gel pieces were sequentially washed 3 times with 25 mM NH4HCO3 and 100% acetonitrile (ACN) to remove excess of detergent and buffer. After the last dehydration in ACN, pieces of gels were rehydrated for 1 h at 4 °C with 2 µL of a 5 µg/mL trypsin proteomic grade solution (Roche) diluted in 25 mM NH4HCO3 in order to ensure sufficient trypsin diffusion and to prevent autocatalysis. Finally, the temperature was raised to 37 °C for an overnight digestion. Peptides were extracted by adding 5 µL of a 1% trifluoroacetic acid (TFA) (v/v)/30% ACN (v/v) solution and vortexing for 30 min. Two µL of the resulting extract was dropped on a 384-600 MTP Anchorchip MALDI target plate (Bruker Daltonic) previously spotted with a 30 mg/mL (w/v) HCCA matrix (Sigma) solution diluted in acetone. After drop drying, each spot was desalted with cold 10 mM ammonium phosphate solution. Protein identification was carried out using MALDI-TOF/TOF instrumentation (Ultraflex II, Bruker Daltonic) in MS and LIFT MS/MS modes. Mass error was fixed at 70 ppm and peptide modifications were assessed as cysteine carbamidomethylation (fixed modification) and methionine oxidation (variable modification). Protein search was performed in NCBI.

Results Isolation of the AOX1- Strain. To inactivate the transcriptional expression of the Aox1 gene, we used the combination of an intron-containing fragment of Aox1 directly linked to its intron-less antisense counterpart. This construction was placed under control of the Aox1 gene promoter to obtain the plasmid Journal of Proteome Research • Vol. 9, No. 6, 2010 2827

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Figure 1. Expression of the AOX protein (Western blot) in 83 and C1 control strains, and in four transgenic clones (T10, 18, 53, and 91) cultivated in TAP NO3 liquid medium under moderate light (50 µE m-2 s-1). In each case, 30 µg of solubilized membrane proteins were loaded on the gel

pULGD22 (see Material and Methods). Cells from the arginineauxotrophic wall-less strain 325 deficient for argininosuccinate lyase were cotransformed with pULGD22 together with the pASL plasmid bearing the Arg7 gene as a selectable marker. Arg+ prototrophic transformants were selected on TAP ammonium agar plates. Assuming that, as observed in tobacco,30 the growth of transformants lacking the AOX1 enzyme is inhibited when the cytochrome pathway of respiration is blocked, 120 arg+ transformants were isolated and tested for their sensitivity to antimycin A (1 µM) + myxothiazol (7.5 µM), two inhibitors of complex III. Spotting cell suspensions on agar plates with or without the inhibitors and incubation for a few days in the light led to the isolation of four transgenic clones (T10, 18, 53, and 91). Clones T18, T53, and T91 could not grow on plates containing the inhibitors, whereas a slight growth was observed for T10. In contrast, the wild-type strain 83 and two arg+ clones (strains C1 and C2) transformed with pASL only were only moderately sensitive to antimycin A and myxothiazol. In the absence of inhibitors, the four transgenic clones grew as control strains, both on TAP NH4 and on TAP NO3 agar medium. PCR amplifications using a primer annealing in the bacterial sequence and another primer in the cDNA sequence of Aox1 showed that at least a fragment of the sense-antisense construct was present in each transgenic strain (data not shown). To verify that the sensitivity to complex III inhibitors was related to a reduced level of AOX1 protein, the four transgenic clones as well as the control strains 83 and C1 were grown in nitrate-containing medium to overexpress AOX1 protein and cell extracts were analyzed by immunoblotting using a polyclonal antibody raised against the C. reinhardtii AOX. As the deduced protein sequences of AOX1 and AOX2 present 57.6% of identity and reveal high similarity for certain segments, a polyclonal antibody has a high probability to recognize both AOX1 and AOX2 proteins.14,15 Figure 1 shows that a strong signal was obtained with the two controls, a much weaker signal with clone T10, whereas no signal at all was detected in transformants T18, T53, and T91. This absence of signal not only supports the fact that AOX1 has been silenced, but also that AOX2 is not expressed significantly in these strains. Clone T53 was conserved for further analyses and called AOX1- strain.

Figure 2. Production of hydrogen peroxide (DAB staining) and superoxide anion (NBT staining) per time unit in control (strain C1) and AOX1- (strain T53) cells incubated in the dark or in the light (50 and 200 µE m-2 s-1). The values obtained for control cells incubated under 50 µE m-2 s-1 light were arbitrarily fixed to 1. The ratios (( standard deviation) between AOX1- and control staining intensities are given in each case.

Functional Assays. To evaluate the impact of AOX1 inactivation on C. reinhardtii cells, several functional assays have been carried out. Cellular respiration in AOX1- strain (T53) was fully sensitive to cyanide and fully insensitive to SHAM (Table 1), suggesting that the alternative pathway does not participate to respiration. Similarly, mitochondrial respiration of AOX1strain exhibits the same behavior (Table 1): fully sensitive to cyanide and insensitive to BHAM. Both immunoblot and respiration experiments strongly suggest that in the AOX1inactivated strain, AOX2 expression does not play a significant role. We have also evaluated the in vivo production of both hydrogen peroxide and superoxide anion in AOX1- and control strains by incubating the cells in the dark or in the light (moderate or high) with DAB and NBT. Exponentially growing cells were used after cultivation in TAP NO3 medium under moderate light. Incubation of the cells in the dark led to a significant oxidation of DAB, reflecting the production of hydrogen peroxide, while no signal was obtained with cells incubated with NBT. Incubation under light favored the accumulation of peroxide and superoxide, especially at high light intensity, pointing out that light-dependent reactions are the major source of ROS. The production of hydrogen peroxide and superoxide anion was higher in AOX1- cells in all experimental conditions (Figure 2). The mean cell volume was about twice higher in transformants compared to control cells and this increase in cell volume was correlated to an increase in protein, chlorophyll, and starch contents (Table 2). At the stationary phase, the total cell volume (the biomass) is the same (around 2.2 mm3/mL) in the wild-type and the AOX1- strain.

Table 1. Mitochondrial and Cellular Dark Respirations (nmol O2 min-1 mg-1 Protein) of Control (Strain C1) and AOX1- (Strain T53) Exponentially Growing Cells Cultivated under 50 µE m-2 s-1 of White Light in TAP NO3 Liquid Mediuma cellular respiration

Total respiration + S/BHAM + S/BHAM + KCN + KCN + KCN + S/BHAM

mitochondrial state III respiration

C1

T53

C1

T53

103 ( 14 97.2 ( 6.1 10.3 ( 7.5 58.5 ( 6.1 7.61 ( 7.42

105 ( 7 105 ( 8 12.2 ( 9.7 8.43 ( 1.65 7.13 ( 7.04

64.2 ( 4.6 62.9 ( 3.5 5.22 ( 0.63 20.3 ( 2.1 7.74 ( 1.47

59.8 ( 4.2 60.5 ( 5.0 3.85 ( 1.52 5.33 ( 0.62 6.64 ( 1.34

a SHAM/BHAM and KCN were used to inhibit AOX and the cytochrome pathway, respectively. The inhibitors were added in the order indicated by the symbol “+”. Values are the means of three independent experiments (( standard deviation).

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Table 2. Cell number/mL, Mean Cell Volume, Protein Content, Chlorophyll Content, And Starch Content of Control (C1) and AOX1(T53) Cells Cultivated in TAP NO3 Medium under 50 µE m-1 s-2 White Lighta strain

growth phase

cell number/mL ( × 106)

cell volume (µm3)

protein content (µg/107cells)

chlorophyll content (µg/107 cells)

starch content (µg/106 cells)

C1

Exponential Stationary Exponential Stationary

3.7-4.4 19-20 2.3-2.7 10.2-13.1

145 ( 5 110 ( 8 286 ( 15 222 ( 19

205 ( 35 128 ( 26 311 ( 9 256 ( 26

27.6 ( 7.1 13.6 ( 0.3 48.3 ( 2.4 25.5 ( 3.6

6.9 ( 2.4 ND 14.6 ( 2.1 ND

T53 a

ND: not determined.

5 are representative examples on both strains. Taking into account the numerous hypothetical proteins (10%) with unknown localization and the contaminants (5%, not from Chlamydomonas origin), we observed around 1% of peroxisomal, 4% of cytoplasmic, and 3% of chloroplastic proteins, and thus, 78% of mitochondrial proteins (Figure 5A). If we exclude the 10% of unknown proteins and the 5% of foreign proteins, we obtain 91% of mitochondrial proteins. Figure 5B classifies the identified mitochondrial proteins according to their function.

Figure 3. Light-saturation curves of photosynthetic oxygen evolution in wild-type and AOX1-silenced cells during exponential phase (3 days) in TAP NO3 medium.

Photosynthetic activities of control and AOX1- strains were also measured during exponential growth on TAP NO3. For this purpose, light-saturation curves of photosynthetic oxygen evolution were determined (Figure 3). When expressed on a chlorophyll basis, oxygen exchange rates were slightly lower in the AOX1- strain, from 100 µE m-2 s-1. This indicates that linear electron transport activity is slightly decreased (not significantly) in AOX1- strains. Effects of Aox1 Silencing on the Mitochondrial and Cellular Soluble Proteomes. To analyze the consequences of AOX1 deficiency (suppression of function) on the metabolic network of the cell, we carried out a comparative study on both the mitochondrial and cellular soluble proteomes from cells in mid log phase of culture (corresponding to (4-6) × 106 cells/ mL and (2-3) × 106 cells/mL for the wild-type and the mutant strain, respectively). 1. Modifications of the Mitochondrial Proteome. A total of 900 protein spots were detected on the 2D gels (Figure 4, pH range 3-11 nonlinear). Ninety of them displayed significant statistical modifications (R ) 0.05, (1.2) in the mutant strain and 82 of them could be identified (Table 3). We also identified 324 protein spots which did not display a significant variation of expression (Table in Supporting Information). To check the purification yield of the mitochondrial extract used for proteomic studies, we have performed 2D-HPLC experiments on Percoll purified mitochondria from both strains. As mitochondria were purified carefully and simultaneously by the same procedure, no difference could be observed between the purity of samples from both origins. Thus, the results given in Figure

Except a few peculiar spots corresponding to subunits of the respiratory chain complexes (two spots over seven for the complex I 49 kDa ND7 subunit, one spot over three for the complex I 51 kDa subunit, one spot over eight for the succinate dehydrogenase subunit A), most proteins of the respiratory chain did not show modification of expression. At the level of ATP synthase, the picture is similar: one spot over three change for the ATP synthase associated 31.2 kDa protein and four spots over 14 for ATP synthase beta subunit (the most important spots do not vary) (see Table 3 and Table in Supporting Information). This indicates that the lack of AOX1 has no impact on the oxidative phosphorylation apparatus. In the AOX1-deficient strain, we found a strong up-regulation of glutathione peroxidase and glutathione reductase (+5 and +8), two of the major ROS scavenging enzymes in the mitochondria, as well as of the cytochrome P450 NO reductase (+2.7) involved in RNS defense. Catalase was, however, found to be weakly down-regulated (two spots over six). This could be due to the extremely strong up-regulation of glutathione reductase and peroxidase that consume H2O2. We also observed an up-regulation (+1.8 in average) of the hybrid cluster protein (HCP), a protein also identified in the mitochondrial compartment by Atteia and co-workers.31 By blasting the protein sequence of HCP, we found an important homology with the hydroxylamine reductase of Syntrophus aciditrophicus. It has been evidenced that HCP acts also as a peroxidase in Escherichia coli,32 highlighting the potentially important role of this protein in ROS scavenging. Several TCA cycle enzymes were down-regulated: citrate synthase (-1.7 in average), isocitrate dehydrogenase NAD-dependent (-1.3), succinyl-CoA ligase (-1.3), oxoglutarate dehydrogenase (-1.3), and pyruvate dehydrogenase (-1.4), suggesting a decrease of Krebs cycle capacity in AOX-silenced mitochondria (Figure 6). Interestingly, these observations are in total opposition with the observed proteomic modifications observed in yeast when AOX was ectopically expressed (creation of function).12 In AOX1- strain, we also observed a strong up-regulation of the mitochondrial carbonic anhydrase (mtCA, +5). This enzyme, which catalyzes the reversible interconversion of soluble carbon dioxide into HCO3-, is only expressed in low CO2 conditions in the mitochondrial compartment.33 Enzymes involved in amino acid metabolism also displayed up- or down-regulation. For instance, the proline oxidase, an enzyme involved in the degraJournal of Proteome Research • Vol. 9, No. 6, 2010 2829

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Figure 4. 2D pattern of the Chlamydomonas mitochondrial proteome: superimposition of wild-type and AOX1- sample images in a 2D gel. In this gel, proteins from wild-type and AOX1- cells are, respectively, labeled with Cy5 and Cy3, so that they, respectively, emit red and green fluorescence. Some proteins (or trains of proteins) are framed in order to give markers of molecular weight (arrows on the vertical dimension) and isoelectric point (arrows on the horizontal dimension). (1) ATP synthase β subunit; (2) ATP synthase 60 kDa subunit; (3) Succinate dehydrogenase subunit A; (4) oxoglutarate dehydrogenase, E1 subunit; (5) Aconitase; (6) Citrate synthase; (7) Isocitrate dehydrogenase NADP-dependent; (8) ATP synthase 31.2 kDa associated subunit; (9) Complex III, Rieske subunit; (10) ATP synthase OSCP subunit; (11) Mitochondrial carbonic anhydrase; (12) Pyruvate dehydrogense E1 subunit, (13) Complex III core subunit 1. See Table 3 or Supporting Information for the regulation values.

dation of proline (antioxidant amino acid) into glutamate, is down-regulated. 2. Modifications of the Cellular Proteome. We undertook a comparative study on the soluble cellular proteome of Chlamydomonas in order to integrate the observed proteomics changes in the mitochondria into a cellular context. Two pH ranges were used to optimize the spot resolution during the first dimension (3-7 NL and 3-11 NL drystrips). The metabolic pathways that are affected at the cellular proteome level in response to AOX1 silencing mainly concern the energy metabolism (Table 4). In AOX1 silenced strain, we detected up-regulation of expression of several enzymes of the pentose phosphate pathway involved in the regeneration of ribulose 1,5-bisphosphate, the key metabolite in the Calvin cycle required for CO2 assimilation. We also highlighted an upregulation of the rubisco activase (+1.5) suggesting a possible increase of CO2 fixation capacity for the synthesis of 3-phosphoglycerate. An up-regulation of the chloroplastic isoforms of the phosphoglycerate kinase (+1.6, +1.3) and glyceraldehyde-3 phosphate dehydrogenase (+1.3) was also observed. The only protein identified in the photosynthetic transport chain that was significantly decreased is the ferredoxin-NADP reductase (-1.3) which catalyzes the reduction of NADPH with electrons from the photosynthetic electron transport chain. At 2830

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the level of glycolysis, pyruvate kinase, which catalyzes the irreversible conversion of phosphoenolpyruvate into pyruvate, is down-regulated in the AOX-silenced strain (-1.4). Moreover pyruvate utilization capacity is also down-regulated. Indeed, we have detected strong down-regulations of pyruvate dehydrogenase, alcool dehydrogenase, acetaldehyde dehydrogenase, Krebs cycle enzymes as well as glyoxylate cycle enzymes. These results suggest an overall decrease in pyruvate consumption capacity for catabolic purposes. The only catabolic pathway that seems to be up-regulated is related to formate production. In Chlamydomonas, formate is mostly produced together with acetyl-CoA during anaerobic fermentation of pyruvate by the pyruvate formate lyase (PFL).34,35 PFL is up-regulated in our analysis, but this enzyme is known to be inactivated by oxygen and requires S-Adenosylmethionine as an activation cofactor.36 The increase in the PFL protein amount could be counterbalanced by the down-regulation of the expression level of the S-Adenosylmethionine synthase (-1.4) which could affect the ability of PFL to consume pyruvate. Altogether, these results indicate an important overall down-regulation of the enzymes of the major catabolic pathways in the cell in addition to those observed into the mitochondria. AOX1 inactivation has also an impact on some anabolic pathways. We detected up-regulations of phosphoglucose isomerase, UDP-glucose 4-epimerase

research articles

Proteomic/Functional Characterization of a C. reinhardtii Mutant a

Table 3. Effects of AOX1 Inactivation on the Mitochondrial Proteome master number

accession number in NCBI

t test

average ratio (AOX1-/WT)

acetyl CoA synthase acetyl CoA synthase aldehyde dehydrogenase aldehyde dehydrogenase aldehyde dehydrogenase aldehyde dehydrogenase

0.017 0.024 1.50 × 10-7 0.00018 0.0014 0.006

TCA Cycle dihydrolipoamide acetyltransferase dihydrolipoyl dehydrogenase dihydrolipoyl dehydrogenase dihydrolipoyl dehydrogenase citrate synthase citrate synthase citrate synthase citrate synthase aconitate hydratase isocitrate dehydrogenase, NAD-dependent isocitrate dehydrogenase, NAD-dependent isocitrate dehydrogenase, NAD-dependent isocitrate dehydrogenase, NADP-dependent isocitrate dehydrogenase, NADP-dependent isocitrate dehydrogenase, NADP-dependent isocitrate dehydrogenase, NADP-dependent succinate-coa ligase beta chain succinate-coa ligase beta chain, minor isoform 2-oxoglutarate dehydrogenase, E1 subunit 2-oxoglutarate dehydrogenase, E1 subunit

protein name

MW

pI

-1.47 -1.74 1.81 1.51 1.94 1.98

74089 74089 58380 58380 58380 58380

7.3 7.3 6.8 6.8 6.7 6.8

0.0038 4.50 × 10-5 0.0083 0.013 0.00025 0.0028 0.0035 0.031 0.037 5.30 × 10-7 0.0021 0.012 0.0013 0.0059 0.014 0.029 0.00062 1.80 × 10-6 0.0087 0.041

-1.59 -1.22 -1.2 -1.24 -1.81 -1.73 -1.64 -1.24 -1.24 -1.41 -1.22 -1.20 1.23 1.23 1.26 -1.25 -1.27 -1.24 -1.33 -1.28

64446 52905 52905 52905 54856 54856 54856 54856 86754 38796 38796 38796 53886 53886 53886 53886 44733 40421 117738 117738

7.8 9.3 9.3 9.3 9.3 9.3 9.3 9.3 8.9 8.8 8.8 8.8 9.5 9.5 9.5 9.5 9.0 7.5 6.8 6.8

10-6 10-6 10-6 10-5

1.42 1.56 1.75 4.66

28193 28193 28193 28193

10.1 10.1 10.1 10.1

Energy Metabolism 337 318 498 492 497 464

XP_001702039

340 613 562 593 640 638 642 651 170 836 831 833 722 715 740 712 751 752 105 71

XP_001696403 XP_001695163

XP_001690955

XP_001695571

XP_001689702 XP_001694857

XP_001698704

XP_001691581 XP_001691582 XP_001692870

1099 1102 1103 1104

XP_001696003

799 796 578 388 622

XP_001697607

307 352 360 367 919

XP_001691632

685 489 511 486 503 500

XP_001695331 XP_001695632

isocitrate lyase malate synthase malate synthase malate synthase malate synthase malate synthase

242 263 234 1211

XP_001689719

655 427 661 630 577

XP_001700272 XP_001694700 XP_001701658 XP_001696763

XP_001702590 XP_001689842 XP_001697021

XP_001693576

mitochondrial mitochondrial mitochondrial mitochondrial

carbonic carbonic carbonic carbonic

Anaplerosis anhydrase, beta type anhydrase, beta type anhydrase, beta type anhydrase, beta type

8.40 1.60 2.10 3.40

× × × ×

Respiratory Chain NADH:ubiquinone oxidoreductase 49 kDa ND7 subunit NADH:ubiquinone oxidoreductase 49 kDa ND7 subunit NADH:ubiquinone oxidoreductase 51 kDa subunit succinate dehydrogenase subunit A ubiquinol:cytochrome c oxidoreductase 50 kDa core 1 subunit

0.0018 0.0054 0.029 0.019 0.0052

1.20 1.25 1.23 -1.25 1.22

53030 53030 52827 69521 55248

8.6 8.6 9.5 6.3 5.9

ATP Synthase beta subunit of mitochondrial ATP synthase beta subunit of mitochondrial ATP synthase beta subunit of mitochondrial ATP synthase beta subunit of mitochondrial ATP synthase mitochondrial F1F0 ATP synthase associated 31.2 kDa protein

0.0013 0.00028 0.0011 0.043 0.00093

-1.21 -1.45 1.30 1.22 -1.24

61954 61954 61954 61954 34111

4.80 4.80 4.80 4.80 7.60

0.01 0.00075 0.006 0.021 0.023 0.038

1.88 -1.83 -1.46 -1.7 -1.57 -1.41

45948 61011 61011 61011 61011 61011

5.9 8.7 8.7 8.7 8.7 8.7

pyruvate-formate lyase pyruvate-formate lyase pyruvate-formate lyase hydroxypyruvate reductase

0.028 0.028 0.038 0.0059

1.57 -1.63 1.30 1.20

91431 91431 91431 45116

6.5 6.5 6.5 9.4

ROS ans RNS Defense Systems cytochrome P450, nitric oxide reductase glutathione reductase glutathione peroxidase catalase/peroxidase catalase/peroxidase

8.40 × 10-6 5.60 × 10-5 1.50 × 10-8 0.00031 0.015

2.68 5.52 7.32 -1.26 -1.34

44185 45630 21499 56407 56407

6.5 8.3 9.8 7.0 7.0

Glyoxylate Cycle

Pyruvate Metabolism

XP_001691480

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Mathy et al.

Table 3. Continued master number

accession number in NCBI

protein name

474 465 466 471

XP_001694671

hybrid-cluster hybrid-cluster hybrid-cluster hybrid-cluster

1001

XP_001701594

426 771 788 753 661 668 735 581 515 847 497 987

XP_001696489 XP_001697168

t test

protein protein protein protein

average ratio (AOX1-/WT)

MW

pI

0.0024 0.003 0.0047 0.042

1.85 1.96 2.04 1.47

70816 70816 70816 70816

8.6 8.6 8.6 8.6

gamma carbonic anhydrase

0.0014

1.25

31320

7.8

Other Functions NADH oxidase putative NADH:flavin oxidoreductase/NADH oxidase putative NADH:flavin oxidoreductase/NADH oxidase putative NADH:flavin oxidoreductase/NADH oxidase cysteine desulfurase cysteine desulfurase dihydropryrimidine dehydrogenase NADP adrenodoxin-like ferredoxin reductase NADP adrenodoxin-like ferredoxin reductase selenophosphate synthase sulfite oxidase voltage-dependent anion-selective channel protein

0.0011 0.0003 0.006 0.013 0.032 0.033 0.022 8.70 × 10-5 0.026 0.0028 0.0014 0.0035

2.00 1.49 1.41 1.27 1.22 1.21 -1.26 1.51 1.24 1.29 1.94 -1.24

64602 41793 41793 41793 50938 50938 41108 55049 55049 31414 64119 28532

9.7 8.7 8.7 8.7 7.0 7.0 9.7 9.9 9.9 5.0 7.9 9.5

Amino Acid Metabolism serine hydroxymethyltransferase serine hydroxymethyltransferase proline oxidase 3-hydroxybutyrate dehydrogenase, mitochondrial precursor 3-hydroxybutyrate dehydrogenase, mitochondrial precursor 3-hydroxybutyrate dehydrogenase, mitochondrial precursor ornithine transaminase alanine aminotransferase alanine aminotransferase

3.40 × 10-5 0.00012 0.00033 7.30 × 10-5 0.0084 0.012 7.00 × 10-5 0.005 0.023

1.58 1.42 -1.42 -1.23 -1.22 1.44 -1.33 1.77 -1.23

57309 57309 61924 28150 28150 28150 48655 58184 58184

9.4 9.4 7.0 9.0 9.0 9.0 6.6 8.6 8.6

Predicted Protein hypothetical protein CHLREDRAFT_188544

3.30 × 10-5

1.53

111622

9.3

pH Regulation

XP_001695008 XP_001699420 XP_001693401 XP_001693400 XP_001701253 XP_001700901

591 595 536 1024 1016 757 719 607 637

XP_001701451

1100

XP_001690485

a

XP_001689646 XP_001700521

XP_001702022 XP_001698518

The master number gives the location of the protein spot in the master gel defined by the DeCyder software.

(converting glucose into galactose), and ADP-glucose pyrophosphorylase (+1.3) which play a key role in starch biosynthesis. Major proteomics modifications are summarized in Figure 7. At last, as it was the case for the mitochondrial proteome, we also detected in the AOX1-silenced strain upregulation of proteins involved in oxidative stress defenses, namely, Fe-SOD or GST transferase (+1.2).

Discussion Here, we report the functional and the proteomic characterization of a C. reinhardtii strain devoid of AOX1. This study offers the opportunity to analyze the phenotypical consequences of Aox1 gene silencing and to estimate the overall metabolic reorganization occurring in response to the lack of the enzyme. It must be pointed out that all the measurements carried out in this study have been performed on cells grown under mixotrophic conditions (acetate supply in the culture medium and permanent light exposure) and using nitrate as a nitrogen source, that is, in conditions of high availability of oxidizable substrate and of maximal AOX1 expression in the wild-type strain. The Alternative Oxidase Plays an Important Role in the Prevention of ROS Production in Chlamydomonas. One of the consequences of AOX1 inactivation was a higher production of hydrogen peroxide and superoxide anion in cells. As mentioned in the Introduction, a major role of AOX in nonthermogenic tissues would be to prevent the over-reduction 2832

Journal of Proteome Research • Vol. 9, No. 6, 2010

of respiratory chain components and the subsequent production of ROS.37 Interestingly, a tobacco cell line deprived of AOX was also found to produce high levels of ROS, whereas in another cell line, overproduction of the enzyme was correlated with a decrease of ROS abundance.38 At the proteomic level, we found in AOX1- strain a strong up-regulation of major enzymes which scavenge superoxide anion and hydrogen peroxide, highlighting the important role of AOX in ROS prevention. However, despite the increase in several scavenging enzymes, we observed a substantially higher production of ROS in AOX1-deficient cells (Figure 2), showing how important is AOX in cell protection. The Absence of the Alternative Oxidase 1 Induces an Important Increase of the Cellular Volume and Biomass in Chlamydomonas. We observed that the absence of AOX1 did not modify the mitochondrial and cellular dark respirations (Table 1). In a transgenic tobacco cell-line, it was similarly observed that the Aox1 gene silencing had no impact on the respiration rates of the cells.30 A remarkable observation in the AOX1-deficient strain was the doubling of the cellular volume and biomass (chlorophyll, protein, and starch content) (Table 2). A similar observation has been reported by Sieger and co-worker39 on AOX-deficient tobacco cells grown under macronutriment limitation corresponding to a higher AOX capacity in control cells. It can be hypothesized that, at the metabolic level, the inactivation of AOX1 could lead to an overproduction of reduced cofactor that

Proteomic/Functional Characterization of a C. reinhardtii Mutant

research articles

could block catabolic reactions and induce a shift toward anabolic reactions. This results in a doubling of cell volume and biomass.

Figure 5. (A) Subcellular distribution of the proteins identified by 2D-HPCL; (B) Classification of mitochondrial proteins according to their function.

The Absence of Alternative Oxidase 1 Is Responsible for a High Mitochondrial Plasticity. It is generally assumed that AOX activity helps to increase the regeneration of oxidized ubiquinone cofactors when required.40 The proteomic response observed at the mitochondrial level in AOX1-deficient cells partially reflects this assumption. Indeed our proteomics data suggest that the capacity of the most important redox cofactors providers is decreased (Figure 6). Hence, in absence of AOX1, it appears that a molecular reorganization occurs to counterbalance the lack of ubiquinol reoxidation. Since no proteomics modification and no change in the respiration were observed, it seems that the OXPHOS apparatus is unaffected by AOX1 ablation. Two strategies were developed by the cell in order to ensure the redox balance of ubiquinone. On the one hand, NADH oxidase, a reduced cofactor consumer not linked to the respiratory chain, is largely up-regulated. Interestingly, NADH oxidase does not require ubiquinone as an electron acceptor but uses water instead, leading to the production of hydrogen peroxide and contributing to the global ROS production. On the other hand, the capacity of the reduced cofactor production in mitochondria seems decreased by down-regulating the overall capacity of the Krebs cycle. Interestingly, a recent study carried out on Arabidopsis thaliana deprived of alternative oxidase has evidenced a decrease of abundance in several intermediary metabolites of the Krebs cycle, strengthening our interpretation of proteomics data.41 This down-regulation of capacity would therefore decrease the level of NADH that feeds the respiratory chain, and would ultimately decrease the level of ubiquinone reduction. Interestingly, we found that ectopic expression of AOX in yeast leads to an opposite proteomic response:12

Figure 6. Scheme summarizing adaptations of the mitochondrial proteome in response to AOX1 inactivation. Regulation levels displayed on the illustration can be found in Table 3. The numbers on the arrows represent the up- or down-regulation of the enzyme catalyzing the reaction. SUCC, succinate; FUM, fumarate; MAL, malate; OAA, oxaloacetate; CIT, citrate; ISO, isocitrate; R-KG, R-ketoglutarate; SUCC-CoA, succinyl-CoA; GSH, reduced glutathione; GSSG, oxidized glutathione; TH, transhydrogenase; ATPs, ATP synthetase. Journal of Proteome Research • Vol. 9, No. 6, 2010 2833

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Mathy et al. a

Table 4. Effects of AOX1 Inactivation on the Cellular Proteome master number

accession number in NCBI

1308* 863 864 881 1228 2080 940* 1101 914* 1011 2023 797

XP_001703643 XP_001689702

1817 1231 1613 1623 1628 1635 1645 1092* 1097* 1711 910 867

XP_001697293 XP_001695632.1 XP_001695331

XP_001700901 XP_001692885 XP_001692395 XM_001701987 XP_001693118.1 XP_001703585

XP_001689719.1

protein name

Mitochondrial Proteins type-II calcium-dependent NADH dehydrogenase aconitate hydratase aconitate hydratase aconitate hydratase aconitate hydratase voltage-dependent anion-selective channel protein NADH:ubiquinone oxidoreductase 76 kDa subunit mitochondrial F1F0 ATP synthase associated 60.6 kDa protein acetyl CoA synthetase acetyl CoA synthetase malate dehydrogenase dual function alcohol dehydrogenase/acetaldehyde dehydrogenase NADPH quinone oxidreductase, mitochondria malate synthase isocitrate lyase isocitrate lyase isocitrate lyase isocitrate lyase isocitrate lyase isocitrate lyase isocitrate lyase isocitrate lyase pyruvate-formate lyase pyruvate-formate lyase

t test

average ratio (AOX1-/WT)

MW

pI

0.033 6.40 × 10-6 0.0064 0.028 0.023 0.023 0.0028 0.049 0.008 3.90 × 10-5 0.018 0.0073

1.33 -1.45 -1.28 -1.27 1.40 -1.76 -1.55 1.33 -1.85 -1.70 -1.25 -1.31

67558 86754 86754 86754 86754 28532 79107 63123 74089 74089 36864 103023

9.0 8.1 8.1 8.1 8.1 9.1 8.1 5.8 7.0 7.0 8.5 7.3

0.026 0.046 0.0014 0.042 0.0036 0.00038 0.002 0.00091 0.01 0.0038 0.014 0.042

1.24 -1.21 -1.30 -1.27 -1.29 -1.28 -1.26 -1.25 -1.20 -1.64 1.36 1.23

36849 61011 45948 45948 45948 45948 45948 45948 45948 45948 91431 91431

5.5 7.7 5.9 5.9 5.9 5.9 5.9 5.9 5.9 5.9 6.5 6.5

0.0041 0.036 0.043

-1.33 1.35 1.27

55233 72336 72336

6.7 7.7 8.0

1.49 1.31 1.30 1.30 1.36 1.24 -1.63 -1.55 1.44 1.50 1.49 1.31 1.63 1.26 -1.35 -1.17 1.53 1.24 -1.33 1.22 1.31 1.58 1.64 -1.28 -1.34

42393 42393 42393 42393 42393 42751 78352 78352 53617 42151 42151 49172 49172 40507 52228 52228 45229 89469 38698 48603 48021 41025 41025 77211 77211

8.6 8.6 8.6 9.6 9.6 8.8 6.9 6.9 5.6 9.0 9.0 8.9 8.9 9.2 6.3 6.3 8.7 5.1 8.5 7.6 6.6 6.0 6.0 5.7 5.7

Glycolysis 1249 1154 1156

XP_001693008.1 XP_001703279.1

1815 1821 1826 1315* 1329* 1809 1021 1024 819* 1273* 1298* 1757 1187 1725 1466 976 1764 1250* 2067 1625 1129* 1760 1777 536* 548*

XP_001691997.1

XP_001690070 XP_001701881 XM_001696828.1 XP_001694038.1 XP_001699523.1 XM_001689819.1 XP_001701593 XP_001692244.1 XP_001697352.1 XP_001698344.1 XP_001701674.1 XP_001702190.1

pyruvate kinase phosphoglucose isomerase phosphoglucose isomerase

Photosynthesis and Pentose Phosphate Pathway sedoheptulose-1,7-bisphosphatase 0.028 sedoheptulose-1,7-bisphosphatase 0.044 sedoheptulose-1,7-bisphosphatase 0.039 sedoheptulose-1,7-bisphosphatase 0.0036 sedoheptulose-1,7-bisphosphatase 0.0032 transaldolase 0.0043 transketolase 0.00025 transketolase 0.0056 6-phosphogluconate dehydrogenase, decarboxylating 0.0011 phosphoribulokinase 0.0014 phosphoribulokinase 0.00029 phosphoglycerate kinase 0.036 phosphoglycerate kinase 0.0024 glyceraldehyde-3-phosphate dehydrogenase 0.012 serine hydroxymethyltransferase 2 0.021 serine hydroxymethyltransferase 2 0.0014 rubisco activase 0.034 rubisco activase 0.00052 ferredoxin-nadp reductase 0.018 low-CO2-inducible protein 0.046 low-CO2 inducible protein 0.027 predicted protein 0.046 predicted protein 0.016 hypothetical protein CHLREDRAFT_82920 0.019 hypothetical protein CHLREDRAFT_82920 0.0032 Sulfate Metabolism

1674 1687

XP_001693343.1

ATP sulfurylase Ats1 ATP sulfurylase Ats1

0.042 0.0094

1.23 -1.48

50490 50490

8.7 8.7

1858 1852

XP_001698672.1

Thiazole Biosynthesis full-length thiazole biosynthetic enzyme full-length thiazole biosynthetic enzyme

0.0057 0.0042

1.33 1.54

37040 37040

6.7 6.7

1927* 1921*

XP_001699262.1 XP_001690591.1

glutathione S-transferase superoxide dismutase [Fe]

0.0054 0.018

1.24 1.26

23922 25899

5.4 9.7

ROS Defense Systems

2834

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research articles

Proteomic/Functional Characterization of a C. reinhardtii Mutant Table 4. Continued master number

accession number in NCBI

657 1181 1182 1077* 704* 1132 1814 1854

XP_001703215.1 XP_001703692

891* 1325 1329 1270 1697 1677 1152* 1093*

XP_001691876.1 XP_001693997

1002* 1325* 1775

XP_001693447.1 XP_001692763 XM_001701662.1

1514 1520 1523 1526

XP_001696661

1046 2128 1930 1256

XP_001698410 XP_001692028 XP_001702364 XP_001689502.1

XM_001691301.1 XP_001692504.1 XP_001697103 XP_001692034 XM_001702433.1

XP_001699068.1 XP_001699763 XP_001695004

average ratio (AOX1-/WT)

MW

0.035 0.0012 0.031 0.0053 0.0024 0.025 0.0017 0.014

-1.25 -1.26 -1.31 -1.25 -1.25 1.20 1.21 1.27

95004 61911 61911 57220 61699 74509 44781 30971

Structural Proteins alpha tubulin 1 beta tubulin 2 beta tubulin 2 beta tubulin 2 actin flagellar associated protein flagellar associated protein flagellar associated protein

0.026 0.013 0.017 0.03 0.044 0.0073 0.022 0.0015

-1.24 -1.49 -1.77 -1.28 -1.34 -1.57 -1.39 -1.29

50182 4.9 50157 4.8 50157 4.8 50157 4.8 42094 5.3 44154 6.5 53056 10.2 53056 10.2

Sugar Metabolism ADP-glucose pyrophosphorylase large subunit UDP-glucose 4-epimerase NAD-dependent epimerase/dehydratase

0.0094 0.00021 0.0032

Adenosylmethionine Synthesis S-Adenosylmethionine synthetase S-Adenosylmethionine synthetase S-Adenosylmethionine synthetase S-Adenosylmethionine synthetase Diverse vacuolar ATP synthase, subunit A plastid lipid associated protein rhodanese-like Ca-sensing receptor predicted protein

protein name

t test

Protein Synthesis, Maintenance, and Folding elongation factor 2 chaperonin 60A chaperonin 60A chaperonin 60C chaperonin 60B2 membrane AAA-metalloprotease peptidyl-prolyl cis-trans isomerase, cyclophilin-type ribosomal protein Sa, component of cytosolic 80S ribosome and 40S

pI

5.6 5.5 5.5 5.4 6.2 5.4 5.1

1.29 1.30 1.33

55675 81408 36568

8.6 5.9 7.1

0.017 0.0073 0.04 0.046

-1.34 -1.53 -1.30 -1.42

43070 43070 43070 43070

6.0 6.0 6.0 6.0

0.022 0.013 0.048 0.046

-1.28 1.25 2.63 1.49

68921 5.7 33144 5.5 38913 9.2 70804 10.0

a The master number gives the location of the protein spot in the master gel defined by the DeCyder software. Master numbers with the symbol “*” and without symbol correspond to protein spots from pH 3-7 NL and pH 3-11 NL pH ranges, respectively.

the introduction of the AOX, decreasing the OXPHOS yield in yeast, promotes catabolic reactions rather than anabolic ones and produces more reducing equivalents in order to maintain a sufficient energy production by the OXPHOS apparatus. The proteomics modifications observed in the Chlamydomonas AOX1- strain can be compared with proteomics data collected on AOX-ablated tobacco cells.39 It has been observed that the expression level of different subunits of the respiratory chain complexes and the cytochromial respiration were decreased, but not the TCA cycle enzymes. Apparently, ablation of AOX has very different effects in both types of cells. However, in tobacco cells, the low dynamic range of the staining dye (Coomassie blue) used to quantify regulation of expression could be a technical limitation compared with 2D-DIGE. The decrease in the TCA cycle capacity in AOX-silenced strain could also be responsible for a decrease in CO2 production and oxaloacetate generation capacities. The strong upregulation of mtCA, that plays a key role in anaplerosis,42 could provide hydrogenocarbonate for the phosphoenolpyruvate carboxylase to produce oxaloacetate, which can be used for amino acid biosynthesis.43 Thus, the up-regulation of mtCA could balance the decrease in the TCA cycle capacity. AOX1 Ablation Leads to an Important Cellular Proteomic Response: A Metabolic Shift toward Anabolism. In our culture conditions, reducing equivalents may be produced by two

distinct ways: either via the photosynthetic electron transport chain or via acetate assimilation. Our functional assays reveal that the rate of photosynthetic linear electron transport is not significantly altered in AOX1- cells, which suggest an unchanged rate of photosynthetic carbon assimilation. In apparent contradiction with this observation, several chloroplastic proteins that belong to the reductive pentose phosphate pathway are strongly up-regulated (Table 4 and Figure 7), which would indicate an increase in the capacity of the Calvin cycle. However, several enzymes of this cycle are known to be lightactivated by the thioredoxin system through reduction of disulfide bridges.44 ROS could therefore have an impact on the activation state of these enzymes in the light through dithiol oxidation. In line with this proposal, it has been evidenced that low concentration of hydrogen peroxide was able to inhibit the activities of several enzymes of the Calvin cycle in higher plants chloroplasts.45 Interestingly, hydrogen peroxide was also found in that study to activate the oxidative part of the pentose phosphate pathway, whereby ribose-5P can be synthesized through oxidative decarboxylation of glucose-6P upon oxidative activation of glucose-6P dehydrogenase. A simultaneous operation of the reductive and oxidative pentose phosphate pathways leads to a futile cycle both in terms of reducing equivalents (successive oxidation and reduction of NAD(P)H) and of carbon budget (successive substrate carboxylation and decarboxylaJournal of Proteome Research • Vol. 9, No. 6, 2010 2835

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Mathy et al.

Figure 7. Scheme summarizing adaptations of the major metabolic pathways in response to AOX1 inactivation. In black, aerobic glycolysis; red, chloroplastic isoforms of glyceraldehyde 3 phosphate dehydrogenase and 3-phosphoglycerate kinase; orange, Krebs cycle; light green, glyoxylate cycle; dark green, pyruvate metabolism; purple, pentose phosphate pathway; light blue, gluconeogenesis. The ratios presented are an average of the different calculated ratios in case of multiple identifications. The numbers on the arrows represent the average ratio between the wild-type and AOX1- strains for the enzyme catalyzing the reaction.

tion). This is usually avoided by thioredoxin-dependent modulation of the activities of enzymes involved in reductive and oxidative branches.46,47 This modulation is potentially affected 2836

Journal of Proteome Research • Vol. 9, No. 6, 2010

by higher peroxide levels in AOX- cells, in such way that increased enzyme levels do not lead to increased CO2 assimilation activity and promote the operation of the above futile

Proteomic/Functional Characterization of a C. reinhardtii Mutant

research articles

Figure 8. Scheme summarizing the effects of AOX1 silencing in Chlamydomonas cells, highlighted by our functional and proteomics studies. Pathways that were found to be up-regulated and down-regulated are colored in red and blue, respectively. UQH2, ubiquinol; ROS, reactive oxygen species; PDH, pyruvate dehydrogenase; Ac-CoAs, acetyl-CoA synthase; mtCA, mitochondrial carbonic anhydrase; OAA, oxaloacetate; ATPs, ATP synthetase; C, cytoplasm; OM, outer membrane; IS, intermembrane space; IM, inner membrane; MM, mitochondrial matrix

cycle. This point would, however, require further investigation since Calvin cycle enzymes from algae were found to be less sensitive to peroxide than higher plant enzymes.48 In our study, the excess glyceraldehyde-3-phosphate generated by the reductive pentose phosphate pathway seems preferentially used for storage, and generation of biomass since the capacity of catabolism in AOX1-silenced strains is depressed. This interpretation is strengthened by our observations of a moderate up-regulation of the UDP-glucose pyrophosphorylase, catalyzing a key step in starch biosynthesis, and of a significantly increased starch/protein or starch/chlorophyll ratio (Tables 2 and 4). A pronounced effect at the proteomic level is also observed in this study on carbon assimilation from acetate. In Chlamydomonas, acetate is converted by the acetyl-CoA synthase and subsequently metabolized through the glyoxylate cycle to produce malate.18 In our analysis, the acetyl-CoA synthase and several enzymes of the glyoxylate cycle are down-regulated per unit of cellular proteins, indicating a decrease of acetate assimilation capacity in AOX1- cells. Important catabolic pathways follow the same general downregulation at the proteomic level, indicating that the capacities of the most important catabolic reactions are slightly depressed in the absence of AOX1 in Chlamydomonas (Figure 7). For instance we have highlighted an important down-regulation of the pyruvate kinase, indicating a probable decrease in pyruvate synthesis capacity and a probable increase in phosphoenol pyruvate availability. A study carried out by Parson and co-workers in tobacco plant deprived of AOX has evidenced a substantial increase of phosphoenol pyruvate derived amino acids (Phe, Trp, Tyr) and a decrease of pyruvate derived amino acids (Ale, Leu, Val)49 which could be the consequence of the down-regulation of the pyruvate kinase.

Conclusion We report here the modifications occurring in Chlamydomonas in response to AOX1 silencing. The basements of this study are the proteomics modifications observed in the mitochon-

drion and in the whole cell. Our data support the idea that the absence of AOX1 favors a metabolic shift toward anabolic reactions by down-regulating the most important catabolic pathway capacities. This proposal is strengthened by the doubling of the cellular volume and biomass. A possible explanation of these events is the following: AOX1 ablation induces a steady-state disequilibrium in the respiratory chain (an excess of reducing equivalent supply with no increase in energetic demand by the cell), with the direct consequence of an over-reduction of the respiratory chain leading to ROS overproduction. In order to escape from this situation, the cell must adapt by (1) increasing the ROS scavenging systems and (2) decreasing the reducing power producers and raising the anabolic reaction capacity to consume reducing power (Figure 8). Thermodynamically speaking, this overall response is a good application of the Le Chatelier principle to biological systems, highlighting the thermodynamical constraints to which a biological system is subjected (high redox potential in this case) and the molecular mechanisms developed by the cell to counteract this constraint.

Acknowledgment. Miche`le Radoux is thanked for technical assistance. This work was supported by grants from the Belgian Fonds de la Recherche Scientifique FNRS (F.R.S.-FNRS 1.C057.09, F.4735.06, FRFC 2.4582.05,), and by Action de Recherche Concerte´e (ARC07/12-04). G.M. is a recipient of a F.R.S.-FNRS doctoral fellowship, P.C. and P.L. are F.R.S.-FNRS research associate, F.F. is a senior research associate of the F.R.S.-FNRS. Supporting Information Available: Master list of proteins. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Jarmuszkiewicz, W.; Czarna, M.; Sluse, F. E. Substrate kinetics of the Acanthamoeba castellanii alternative oxidase and the effects of GMP. Biochim. Biophys. Acta 2005, 1708 (1), 71–78.

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