The Thylakoid Membrane Proteome of Two Marine Diatoms Outlines

ARTICLE pubs.acs.org/jpr

The Thylakoid Membrane Proteome of Two Marine Diatoms Outlines Both Diatom-Specific and Species-Specific Features of the Photosynthetic Machinery Irina Grouneva,† Anne Rokka,‡ and Eva-Mari Aro*,† † ‡

Department of Biochemistry and Food Chemistry, Molecular Plant Biology, Tykist€okatu 6A, FI-20520, University of Turku, Finland Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Tykist€okatu 6A, FI-20520, Turku, Finland

bS Supporting Information ABSTRACT: The thylakoid membrane of photoautotrophic organisms contains the main components of the photosynthetic electron transport chain. Detailed proteome maps of the thylakoid protein complexes of two marine diatoms, Thalassiosira pseudonana and Phaeodactylum tricornutum, were created by means of two-dimensional blue native (BN)/SDS-PAGE coupled with mass spectrometry analysis. One novel diatomspecific photosystem I (PS I)-associated protein was identified. A second plastid-targeted protein with possible PS I interaction was discovered to be restricted to the centric diatom species T. pseudonana. PGR5/PGRL homologues were found to be the only protein components of PS I-mediated cyclic electron transport common to both species. For the first time, evidence for a possible PS I localization of LI818-like light harvesting proteins (Lhcx) is presented. This study also advances the current knowledge on the light harvesting antenna composition and Lhcx expression in T. pseudonana on the protein level and presents details on the molecular distribution of Lhcx in diatoms. Above mentioned proteins and several others with unknown function provide a broad basis for further mutagenesis analysis, aiming toward further understanding of the composition and function of the photosynthetic apparatus of diatoms. The proteomics approach of this study further served as a tool to confirm and improve genome-derived protein models. KEYWORDS: BN, diatoms, LI818-like light harvesting proteins, PS I-mediated cyclic electron transport, thylakoids

’ INTRODUCTION Diatoms (Bacillariophyceae) are unicellular, eukaryotic algae with a wide distribution in sea and fresh water. They belong to the group of the Heterokontophyta and have considerable ecological significance, estimated to be responsible for up to one-third of carbon sequestration in the oceans.1
3 Diatoms originate from a secondary endosymbiosis event between a photosynthetic red algae ancestor and a heterotrophic eukaryote.4 As a consequence, diatom chloroplasts possess four envelope membranes instead of two. Also, the thylakoid organization as well as the composition of the pigment
protein complexes differ from those in other algae groups and higher plants. Diatom thylakoids, the site of light harvesting and the photosynthetic electron transport chain, are organized in bands of three with no differentiation into stacked and unstacked regions and apparently also lacking the spatial segregation between photosystem (PS) II and PS I.5 This places diatoms' thylakoid organization close to brown algae (thylakoid lamellae bands of three6), but clearly sets it apart from green algae and higher plants with a spatial segregation of PS II and PS I into distinct grana and stroma lamellae regions, respectively.7 Such segregation of photosystems requires specific regulatory mechanisms to guarantee equal distribution of excitation energy to both r 2011 American Chemical Society

photosystems under naturally fluctuating light intensities.8,9 This occurs via rapid reversible phosphorylation-triggered10,11 redistribution of light harvesting antennae between PS II and PS I, also called state transitions. This process is present in green algae, higher plants, and red algae,12 but not in diatoms.13 This raises the possibility for the existence of specific regulatory mechanisms for excitation energy distribution between the two photosystems in diatoms. In recent years, considerable progress has been made in the understanding of the molecular organization of the light-harvesting complexes, called fucoxanthin-chlorophyll a/c-binding proteins (FCP) in diatoms,14
17 and, to a lesser degree, the main photosynthetic components PS I and PS II.16,18 There are three different subtypes of FCPs. One of them is most likely involved in photoprotection instead of light harvesting. A precise characterization of FCP association with either the PS I/PS II core or the peripheral light harvesting antennae is therefore of the utmost importance toward the elucidation of their specific functions. There are considerable gaps in the current knowledge of the overall composition of the thylakoid protein complexes in Received: June 21, 2011 Published: October 21, 2011 5338

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Journal of Proteome Research diatoms. Of individual complexes, particularly little information is available on the diatom PS I. Besides its role in the linear electron transport and the generation of reducing equivalents in the form of NADPH, PS I is also the site of cyclic electron transport (CET). PS I-mediated CET was shown to be of considerable importance for photosynthesis in general.19 It provides an opportunity for adjusting and regulating the production of ATP and NADPH according to metabolic demand. In cyanobacteria, there is evidence of a connection between CET and carbon concentrating mechanisms (CCM).20,21 The latter are widespread among aquatic species, due to the low CO2 concentrations in water. This is the reason why low CO2inducible proteins are of considerable interest in connection with algal photosynthesis. The targeted diatom species of this study, the centric diatom Thalassiosira pseudonana and the pennate diatom Phaeodactylum tricornutum, were chosen because their full genome sequences are available.22,23 This is a prerequisite for wide mass spectrometry (MS)-based proteome analysis. The two diatoms used in this study share around 57% of their genes and approximately 1328 of those common genes are unique to diatoms (BLASTP expected cut off value of 10
5).23 In addition, each of these two diatoms possesses around 4000 genes (3912 in T. pseudonana) that are unique to the given species. When T. pseudonana was first sequenced in 2004, around half of the predicted genes could not be assigned a function based on sequence homology.22 Although 86% of predicted genes have expressed sequence tags (EST, gene expression under 16 different conditions23), a reliable verification of protein models can only come from proteomics data. Two recent studies employed proteomic analysis on whole T. pseudonana cells,24,25 advancing the field considerably. The present proteomics study is the first to focus particularly on the photosynthetic electron transport chain organization. A comparative study between two diatoms was chosen because of the vast genetic and physiological variability within the group and also as an attempt to elucidate species-specific features. The main goals were to investigate the overall subunit composition of thylakoid protein complexes, to extend the current knowledge on FCP distribution between PS I and the major light harvesting complexes in T. pseudonana as well as an extensive search for components of the PS I-mediated CET in diatoms. On the physiological level, some major differences regarding the mechanism of PS I-mediated CET have already been observed between P. tricornutum and a centric diatom, Cyclotella meneghiniana.26 However, the molecular mechanisms responsible remained a matter of speculation, since these can differ between photosynthetic organisms. Thus, addressing the protein background of PS I-mediated CET in diatoms, a highly successful group of marine algae, was of considerable interest as well.

’ EXPERIMENTAL SECTION Culture Growth

Two diatoms, T. pseudonana CCMP1335 (obtained at the Provasoli-Guillard National Centre for Culture of Marine Phytoplankton) and P. tricornutum CCAP 1055/1 (obtained at the Scottish Marine institute Culture Collection of Algae and Protozoa), were grown as batch cultures at 15 °C in a light-dark regime of 14
10 h in f/2 medium supplemented with vitamins.27,28 The growth light intensity was 50 μmol photons m
2 s
1. In the case of T. pseudonana, the growth medium was

ARTICLE

supplemented with 1.06  10
4 M Na2SiO3. T. pseudonana was additionally kept as air-lift cultures (CO2-replete). Isolation of Thylakoid-Enriched Organelle Membrane Fraction

Diatom cells were harvested from standard growth light conditions (1 h after turning the lights on, if not stated otherwise) by centrifugation at 4000g for 10 min. The cell pellet was washed once and resuspended in 15 mL of isolation buffer A (10 mM Mes, 2 mM KCl, 5 mM EDTA, 1 M sorbitol, pH 6.5). The cells were passed through a French press two times at 11 000 psi and the flow-through was centrifugated at 1000g for 10 min in order to remove whole cells, debris, and silica shells from the lysate. The supernatant was centrifugated at 20000g for 20 min and then washed twice in washing buffer B (10 mM Mes, 2 mM KCl, 5 mM EDTA, pH 6.5). Protein content of the membrane pellet was measured according to Lowry29 and 100 μg of protein was loaded per well for blue native (BN) gel electrophoresis. Centrifugation at 20000g led to a lower yield but higher purity of thylakoid fractions and less mitochondrial contamination as compared to centrifugation at 40000g.14,17 Solubilisation of Membrane Fraction

Pelleted membranes were washed once, resuspended in 25BTH20G buffer (25 mM BisTris-HCl/20% glycerol, pH 7) to a protein concentration of 10 μg μL
1, and then supplemented with either 4%, 2%, or 1% n-dodecyl β-D-malatoside (DM, qw/v) at a ratio of 1:1 (v/v). This yielded a final protein content of 5 μg μL
1 and final DM concentrations of 2%, 1%, or 0.5%. This corresponds to a detergent to protein ratio of 4, 2, and 1 (w/w), accordingly. Solubilization was carried out at 4 °C for 20 min. Unsolubilized particles were removed by centrifugation at 10000g for 30 min. The supernatant was loaded onto a BN gel. 2D BN/SDS-PAGE

Thylakoid membranes from two diatoms, T. pseudonana and P. tricornutum, were subjected to two-dimensional (2D) gel electrophoresis, a BN-PAGE in the first dimension and a denaturating SDS-PAGE in the second. BN gel electrophoresis was carried out as described previously,30 with a continuous acrylamide gradient between 5% and 12.5%. In addition, large pore BN gels, optimized for thylakoid protein complexes,30 were also used to ensure an optimal separation of putative protein complexes bigger than PS I. Lanes were excised from BN gels and subjected to in-gel equilibration in Laemmli buffer supplemented with 10% beta-mercaptoethanol for 1.5 h. The strips were subsequently loaded on top of either 15% or 12% acrylamide gels containing 6 M urea and 20% SDS and ran overnight. Gels were then stained nonquantitatively with silver nitrate according to Blum et al.31 Material for Mass Spectrometric Protein Analysis

MS-based protein identification was first carried out from single protein spots in 2D gels of T. pseudonana. On the basis of these results, complementary and unique protein spots were analyzed from 2D gels of P. tricornutum as well. In addition to single 2D protein spots, entire bands of protein complexes were excised directly from BN gels and subjected to MS analysis. At least three separate membrane preparations were separated by BN-PAGE and analyzed by MS. In order to characterize the distribution of Lhcx proteins, two preparations from cells exposed to 8 h high light (HL, 700 μmol photons m
2 s
1) were also analyzed for T. pseudonana. Although the separation between complexes by BN electrophoresis was found to be of reasonable resolution, a carry-over of

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Table 1. Proteins Identified by MS in Thylakoid Membranes from T. pseudonana and P. tricornutuma peptide sequence/peptide protein ID sample origin (NCBI)

protein description

Mascot number of number of protein sequence scoreb matched peptides samplesc coverage [%]

E-value for peptide

score (>29 means identity/extensive homology)

T. pseudonana PS I (1)

118411096 PsaA

614

15

5

22

(1)

118411153 PsaD

546

10

5

70

(1)

118411097 PsaB

471

15

5

24

(1) (1)

118411182 PsaC 118411168 PsaF

196 194

4 5

5 5

54 38

(1)

118411163 PsaL

166

5

5

32

(1)

223993351 20965

76

2

5

14

(1)

223995405 268304

264

2

5

26

0.052

VLGPIMSK (31)

2.2  10
6

EPGAIENGSWVR (73)

1.6  10
8

NSYVPATGGDGGQG QFGAQSPNDWR (92)

1.8  10
10

IEEFAAEKPYGFTSSDAAMEELVGK (111)

(1) lumend

224013736 predicted,

(1)

145

3

2

14

224006786 Lhcr1

607

5

5

24

(1)

224000649 Lhcr4

518

7

5

36

(1)

224001216 Lhcr10

496

6

5

32

(1)

224001956 Lhcr3

380

6

5

40

(1)

224006233 predicted

329

7

5

46

(1) (1)

224011689 Lhcf10 224015308 PS I LHC

305 300

6 5

2 5

40 57

(1)

223999739 Lhcr14

270

4

5

24

(1)

224015330 hypothetical

253

6

5

62

(1)

223997688 Lhcr7

219

4

5

22

(1)

223997118 Lhcf7

175

4

5

25

(1)

223995135 Lhcr11

174

5

5

15

(1)d

224013064 Lhcx2

348

5

1

40

(1)

224013212 Lhcx1 224011607 Lhcx6_1

130

3

2

25

(1)d

224002166 Lhcx4

129

2

2

14

(2 + 3)

193735617 PsbC

2492

17

5

36

(2 + 3)

118411113 PsbB

2421

22

5

36

(2 + 3)

118411148 PsbD

2162

9

5

29

(2 + 3)

118411180 PsbA

1548

10

5

25

(2 + 3) (2 + 3)

118411160 PsbE 118411116 PsbH

256 343

3 1

5 5

41 21 54

HL-induced

PS II

3  10
5

LGEILRPLNAEYGK (61)

Cyt b6/f (4)

118411137 PetA

3020

12

5

(4)

223995629 Predicted, (Fe
S)

2099

10

5

58

(4)

118411154 PetB

1782

5

5

28

118411155 PetD

97

2

5

13

223993505 Lhcf9

2945

7

2

28

224015764 PetB (4) FCP oligo (5)

224000930 Lhcf8 (5)

223997118 Lhcf7

304

5

1

28

(5)

224012385 Lhcf4

182

4

1

14

(5)

224012180 Lhcf2

177

4

1

15

224012869 Lhcf1 5340

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Table 1. Continued peptide sequence/peptide protein ID

protein

Mascot

number of

(NCBI)

description

scoreb

matched peptides

samplesc

coverage [%]

224011910 Lhcf5

167

4

1

16

224012180 Lhcf2

6615

8

5

46

sample origin (5)

number of protein sequence

E-value for

score (>29 means

peptide

identity/extensive homology)

FCP trimer (6)

224012869 Lhcf1 (6)

224011910 Lhcf5

4457

9

5

60

(6)

223993505 Lhcf9

2756

7

5

45

(6)

224000930 Lhcf8 224012725 predicted

1675

7

5

43

(6)

223998112 Lhcf6

1521

11

5

53

(6)

224012385 Lhcf4

1113

6

5

31

(6)

223997118 Lhcf7

189

5

5

39

(6)

224011607 Lhcx6_1

106

3

2

16

(6)d

224013064 Lhcx2

179

2

2

18

164

4

2

22

224013212 Lhcx1 (6)d free protein

224002166 Lhcx4

(7) lumen

224003107 PsbO, precursor

846

14

3

63

(7) lumen

118411100 PsbV

498

9

3

68 38

(7) lumen

223993775 Predicted, PsbQ’

257

10

2

(7) lumen

224009442 Hypothetical, Psb31

191

5

2

39

(7) lumen

223996703 Predicted, PsbU

181

5

3

28

(7) lumen

224006027 Hypothetical, Cyt c6

82

2

2

15

(7)

223999427 Hypothetical, PGRL

(7)

223999569 Predicted, PGR5

(7)

223999351 thylakoidal processing

(7)d

224013064 Lhcx2

134

5

4

15

83

2

3

14

141

5

3

23

2601

7

5

43

9.5  10
5

EALEQYLAGGR (57)

0.011

NAMPAFGGR (37)

1.2  10
5

FGIFSPAVYVAK (66)

0.0026

IGLGNDR (44)

peptidase 224013212 Lhcx1 (7)d (7)

224013062 Lhcx6 224011607 Lhcx6_1

243 224

5 6

2 4

29 39

(7)d

224002166 Lhcx4

215

5

2

22

(7)d

223994783 Lhcx5

193

4

4

14

(7)

223996699 Lhcf11

1383

6

2

43

(2)/(3)

223992735 predicted, low

469

12

4

16

(3)

223999673 predicted, low

328

7

4

21

118411112 AtpA

499

13

3

26

118411134 AtpB

305

10

3

21

118411109 AtpG

192

4

2

27

118411110 AtpF

165

7

2

36

(1) (1)

219115657 Lhcr1 219124057 Lhcr13

401 479

4 6

2 2

28 36

(1)

219122943 47485

580

4

2

33

(1)

219112535 Lhcr3

400

5

2

43

(1)

219110739 Lhl1 (early light

247

7

2

29

CO2-inducible CO2-inducible plastid ATPase

P. tricornutum PS I

induced) 5341

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Table 1. Continued peptide sequence/peptide protein ID

protein

Mascot

number of

sample origin

(NCBI)

description

scoreb

matched peptides

samplesc

coverage [%]

(1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) PS II mono (3)

219123709 219125332 219111267 219126416 219116130 219129158 219110211 219127823 219109965 219126740 219117950

358 365 453 287 173 231 180 223 76 291 91

4 6 5 4 3 3 3 3 2 2 2

2 2 2 2 2 2 2 2 2 2 1

33 34 39 31 10 22 23 15 9 13 13

62

2

1

6

Lhcr14 Lhcf8 Lhcr4 48798 Lhcf16 Lhcf17 Lhcr12 Lhcr11 17531 Lhcr2 Lhcx1

219119979 Predicted, low CO2-inducible

number of protein sequence

E-value for

score (>29 means

peptide

identity/extensive homology)

0.0052

LLTSPVPLFYSR (39)

0.00052

LPAPQAQAIIDAAHRP NR (48)

FCP trimer (6) (6) (6) (6) (6) (6) (6) (6) (6) free protein (7) (7) (7)

219112233 219112235 219128858 219123828 219128856 219112237 219125332 219112239 219129163 219126950

Lhcf4 Lhcf3 Lhcf5 Lhcf10 Lhcf11 Lhcf2 Lhcf8 Lhcf1 Lhcf12 Lhcf9

219117950 Lhcx1 219110471 Lhcx2 219110655 predicted, PGRL

7348

10

2

56

4153 3804 3524 2802 2752 2377 1878 214

5 10 10 6 11 7 3 5

2 2 2 2 2 2 2 2

44 55 44 36 56 31 19 31

754 346 224

7 5 7

2 1 1

54 49 21

a

Proteins were identified once from a single spot in a 2D gel and multiple times from bands cut from BN gels. Proteins are grouped according to their affiliation with a protein complex and the numbers in brackets refer to the complexes in Figure 1A. The identity level scores given for proteins and peptides represent the highest score acquired for a protein/treatment, not mean values. Scores below 100 belong to short polypeptide sequences, which however were identified multiple times and the spectrum was verified manually. See Supporting Information for delta (Δ) values and ion series of individual peptides. The products of the gene pairs Lhcf9/Lhcf8, Lhcx1/Lhcx2, and Lhcf1/Lhcf2 are not able to be distinguished by MS analysis because the mature proteins are either identical or produce the same set of peptides in LC MS analysis. Therefore, there are two DB entries given but only one set of protein data. Sequence coverage percentage is based on precursor protein DB entries, including target sequences, not the mature protein as found in the chloroplast. This means that MS coverage values for nucleus-encoded proteins are underestimated. Proteins belonging to PS II were identified both in the monomer and the dimer complexes. b Overall score for entire protein. c Number of samples containing a given protein, taken both from complex BN gel samples and identified as a single spot in 2D gels. d See text for pretreatment conditions.

proteins cannot be fully prevented in the case of bands excised directly from BN gels. Nevertheless, the verification of these results came from single 2D gel protein spots, where unequivocal protein identification was possible in the majority of cases. The affiliation of a given protein to a particular complex, as shown in Table 1, was further based on (1) the known function derived from sequence homology to other species, (2) a logical exclusion of proteins overlapping with other bands, for example, verification of PS II subunits by excluding PS I proteins overlapping with PS II dimer and exclusion of Cytb6/f proteins overlapping with PS II monomer in the first dimension, and (3) high scores and sequence coverage as compared to contaminants. In addition, it has to be stressed that the proteins listed as putative novel PS I

subunits in Table 1 were exclusively identified in band 1 (PS I, see Figure 1A). It also has to be stated that MS analysis of FCP samples was always carried out separately in order to avoid contamination of PS I fractions with peripheral antenna proteins. Protein spots or native protein complexes were excised from silver-stained gels and subjected to an in-gel trypsin digestion.32 The resulting peptides were extracted with 50% acetonitrile/5% formic acid and dried in a vacuum centrifuge. Prior to measurements, dried peptides were dissolved in 2% formic acid. Mass Spectrometry

The LC
MS/MS analysis was performed on a nanoflow HPLC system (Ultimate 3000, Dionex, Sunnyvale, CA) coupled 5342

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Figure 1. Main thylakoid protein complexes of T. pseudonana, separated by BN-PAGE (A) and a schematic distribution of LI818-like proteins in LLand HL-treated T. pseudonana cells (B). The gel was partially destained for clearer visibility. According to size, the protein complexes from top to bottom are (1) PS I, (2) PS II dimer, (3) PS II monomer, (4) cytochrome b6/f, (5) oligomeric FCPs, (6) trimeric FCPs, and (7) free proteins.

to a QSTAR Elite mass spectrometer (Applied Biosystems/MDS Sciex, Canada) equipped with a nanoelectrospray ionization source (Proxeon, Odense, Denmark). Peptides were first loaded on a trapping column (0.3  5 mm PepMap C18, LC Packings) and subsequently separated inline on a 15 cm C18 column (75 μm  15 cm, Magic 5 μm 200 Å C18, Michrom BioResources Inc., Sacramento, CA, USA). The mobile phase consisted of water/acetonitrile (98:2 (v/v)) with 0.2% formic acid (solvent A) or acetonitrile/water (95:5 (v/v)) with 0.2% formic acid (solvent B). A linear 14 min gradient from 2% to 35% B was used to elute peptides. For complex samples, a 24 min gradient was tested as well, but no major differences were detected. MS data was acquired automatically using Analyst QS 2.0 software (Applied Biosystems/MDS Sciex, Canada). An information dependent acquisition method consisted of a 1-s TOF MS survey scan of mass range 350
1500 m/z and two product ion scans of mass range 50
2000 m/z. Two most intense ions over 20 counts, with charge state 2
4 were selected for fragmentation. Once an ion was selected for MS/MS fragmentation, it was put on an exclusion list for 60 s. Curtain gas was set at 20 L/min, nitrogen was used as the collision gas, and the used ionization voltage was 2300 V.

For Mascot, DB search settings included precursor-ion mass tolerance of 0.2 Da, fragment-ion mass tolerance of 0.2 Da, one missed trypsin cleavage, variable modifications of carbamidomethylation of cysteine and methionine oxidation. A significance threshold of p < 0.05 was used. For protein scores below 100, a manual verification of actually matched ion series was performed. This was necessary for short protein sequences with single peptide hits. The protein scores given for a particular protein are representative not mean values. In the case of single peptide identification or unknown proteins, detailed information on single peptides was included.

Protein Database Search

’ RESULTS

Peak list files were created by Analyst QS 2.0. Database (DB) searches were performed by Mascot (v. 2.2.06) against a DB containing 20545 T. pseudonana and P. tricornutum protein sequences downloaded from NCBI (http://www.ncbi.nlm.nih.gov/entrez/ query.fcgi?db=Protein&cmd=Search&dopt=DocSum&term= txid296543, accessed April 4, 2011) and also a number of common laboratory contaminants (downloaded from ftp:// ftp.thegpm.org/fasta/cRAP). In order to verify results, test searches were also run against the original diatom genome project JGI DB (containing 22 267 protein sequences from both chromosomes and unmapped sequences downloaded from http://genome.jgi-psf.org/Thaps3/Thaps3.download.ftp. html and http://genome.jgi-psf.org/Phatr2/Phatr2.download. ftp.html, accordingly).

BLAST Searches and Functional Annotation

Proteins designated as “predicted”, “putative”, or “hypothetical” in the DB were subjected to a sequence similarity search (NCBI protein BLAST tool) in order to establish their possible function. Putative protein size is given as calculated from DB entries, including target signal sequences. Nucleus-encoded mature proteins are therefore expected to be slightly smaller in size. The heterokont-specific online tool HECTAR was used for protein plastid localization prediction.33 Possible membrane proteins were identified using the SOSUI system for prediction of membrane/soluble proteins and transmembrane helices.34

1. Macromolecular Protein Complexes of the Thylakoid Membrane

BN gel electrophoresis of thylakoid membranes from the centric diatom T. pseudonana resulted in a reproducible pattern of protein complexes including (1) a broad band of PS I complexes, (2) a PS II dimer, (3) a PS II monomer, (4) the Cytb6/f complex, partially overlapping with PS II monomer, (5) plastid ATP synthase, (6) two brown FCP bands of different protein composition, and (7) a free protein band, consisting of proteins detached from the membrane during detergent solubilization or not associated with any of the bigger complexes. Figure 1A shows an example of a BN gel from T. pseudonana. The present study once more confirmed that in diatoms PS I occurs as 5343

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Figure 2. Overview of T. pseudonana (A) and P. tricornutum (B) thylakoid protein composition resolved by 2D BN/SDS-PAGE. One asterisk in panel C marks a putative PS I-associated T. pseudonana-specific protein (gi|223995405 in Table 1). The same protein spot also contained Lhcx6_1. Three asterisks in panels A and E designate a putative low-molecular novel PS I protein, present in both T. pseudonana (gi|223993351) and P. tricornutum (gi|219110593). Panel E shows the lower molecular region of panel B (indicated by arrow). Panel D shows the major PS II proteins from a CO2-replete T. pseudonana culture lacking a putative low-CO2-inducible protein (gi|223992735) designated by two asterisks in panel A. SDS-PAGE acrylamide concentration of the separation gel was 12% in A and 15% in B.

a monomer.15,16 However, there were obvious variations in size between PS I complexes. These appeared to be due to a variable amount of bound FCP (see Figure 2). Suggestions that PS II forms dimers in diatoms35 were also confirmed, since PS II could be detected both as a monomer and a dimer after 2% and 4% DM solubilization in this study. For P. tricornutum very similar results were obtained, with the exception of FCP protein composition (see below) and the proteins occurring specifically only in T. pseudonana. With the exception of HL treatment, cells were harvested from standard growth conditions (50 μmol photons m
2 s
1) because earlier reports indicated highest photosynthetic activity in PS II particles isolated from LL adapted cells.27 Results shown in Figures 1 and 2 were obtained from batch cultures (CO2-limited) of T. pseudonana.

2. Subunit Composition of the Thylakoid Protein Complexes

Membrane protein complexes separated by BN gels were subjected to a subsequent denaturating treatment and SDS
PAGE separation in the second dimension. In this way, the subunit composition of protein complexes was made visible. Figure 2 shows 2D BN/SDS
PAGE images of thylakoid membrane preparations from T. pseudonana (Figure 2A) and P. tricornutum (Figure 2B). Table 1 lists the protein composition of the main diatom photosynthetic complexes derived both directly from BN gels (entire protein complex) and from 2D gels (single protein spots) and subsequently identified by LC
ESI
MS/MS analysis. Listed proteins were identified at least once as a spot from 2D BN/SDS gels and multiple times from complex protein bands cut 5344

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Figure 3. Amino acid sequence alignment between a putative novel PS I-associated protein below 10 kDa in T. pseudonana (gi|223993351) and its closest homologue in P. tricornutum (gi|219110593). The protein was identified from PS I lanes as a nucleus-encoded putative sequence.

directly from BN gels. The proteins were grouped according to their position in BN gels and therefore according to their association with a given multisubunit complex. Thylakoid lumen proteins were mostly deduced based on the presence of the double arginine motif (RR), a marker of the ΔpH-dependent protein transport system, in combination with a putative cleavage site for the thylakoidal processing peptidase (containing the motif A-X-A). One notable exception was cytochrome c6, which is transported across the thylakoid membrane by the Sec-dependent pathway and therefore lacks the RR motif (reviewed in Mori and Cline36). 3. Photosystem I

The following plastid-encoded proteins were identified as part of the T. pseudonana PS I complex (Figures 1 and 2A): PsaA, PsaB, PsaF, PsaL, the PS I iron
sulfur center PsaC, and the PS I ferredoxin-binding subunit PsaD. The precise position of PsaF in 2D gels could not be established unequivocally due to multiple hits for this protein throughout the 20
15 kDa region in the second dimension. In addition to the above plastid-encoded proteins, two putative nucleus-encoded proteins appeared to be associated with PS I in T. pseudonana as well. Both were recognizable in the PS I lane of 2D gels (Figure 2A,C) and possess clear plastid target signals (PTS). The first protein (gi|223993351) was below 10 kDa, based on its position in the gel. It has a close homologue in P. tricornutum (Figure 2E). Figure 3 shows the sequence alignment between the two proteins from T. pseudonana and P. tricornutum. The P. tricornutum protein had the same position in the gel as T. pseudonana despite a DB entry of a considerably longer protein in the pennate diatom. No possible function for those putative proteins could be predicted by a wide BLAST homology search. There were no close matches in the DB beyond the two diatom proteins shown here. The second protein (gi|223995405) was found to be T. pseudonana-specific (no homologue in P. tricornutum and no significant match in any other organism) and estimated to be approximately 20 kDa in size. This protein appeared to be unique to centric diatoms and no prediction of its function could be made. Its position in the 2D gel overlapped with Lhcx6_1 (Figure 2C). See Supporting Information for MS sequence coverage. A third novel protein (gi|224013736) detected in PS I bands from BN gels was only present in samples pretreated with HL (700 μmol photons m
2 s
1, 8 h).

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A number of FCP proteins specific for PS I were detected in T. pseudonana (see Table 1). These differed clearly from FCPs in the free FCP bands, which represented the peripheral antennae believed to be common to PS II and PS I. The PS I-specific antenna was comprised mainly of Lhcr proteins, while the detached antenna was exclusively made up of Lhcf proteins. Most intriguingly, LI818-like proteins were identified as part of PS I in this study. Lhcx6_1 was detected in the PS I band of T. pseudonana under standard LL growth conditions. Upon HL exposure (700 μmol photons m
2 s
1, 8 h) one additional Lhcx protein, Lhcx4, appeared in PS I bands taken from BN gels. Lhcx 1/2 was also detected as part of the PS I band in T. pseudonana cells kept in low silica medium for five days (Table 1). In P. tricornutum, Lhcx1 was detected as part of PS I under LL. This is the first indication that LI818-like proteins bind to PS I in diatoms. 4. Photosystem II

Two lanes were identified as PS II in T. pseudonana thylakoids subjected to BN/SDS-PAGE, showing the presence of PS II dimers alongside PS II monomers over the range of detergent concentrations used in this study (2
4% DM). Protein association with PS II therefore could be easily recognized by this double lane pattern in the second dimension (Figure 2A,B). The main PS II proteins identified were D1 (PsbA), D2 (PsbD), CP43 (PsbC), CP47 (PsbB), cytochrome b559 alpha chain (PsbE α), and the 10 kDa phosphoprotein (PsbH) subunit. It is important to emphasize that PS II complexes obtained in this study lacked the oxygen evolving complex (OEC). PsbO, PsbV, PsbU, PsbQ0 , and the diatom-specific subunit Psb31 were instead identified in the free protein fraction (see below) following 2% DM solubilization, which indicates that the OEC was detached during the detergent treatment commonly used in this study. 5. Cytochrome b6/f and ATP Synthase

Three protein spots belonging to the Cytb6/f complex (band 4 in Figure 1) were clearly visible in 2D gels (Figure 2A,B). They were identified to be the subunits Cytf (PetA), Cytb6 (PetB), overlapping with the Fe
S domain containing Rieske protein in Figure 2A, and subunit IV (PetD). The Rieske protein identified on the protein level in this study is nucleus-encoded, possesses a PTS, and is designated as “predicted” in the DB. Four plastid-encoded subunits of the chloroplast ATP synthase (AtpA, AtpB, AtpG, and AtpF) were identified. AtpA and AtpB belong to the CF1 part of the multisubunit complex.37,38 They were accordingly predicted to be soluble proteins (SOSUI system for prediction of membrane proteins)34 and were found in the lane overlapping with oligomeric FCPs. AtpG and AtpF were identified in the PS II monomer/Cytb6/f lane and belong to the ATPase part designated as CFo which is embedded in the thylakoid membrane. AtpG was predicted to have one transmembrane helix.34 These results suggest that CF1 and CFo dissociate during membrane solubilization. 6. Fucoxanthin-Chlorophyll a/c-Binding Proteins

Table 1 summarizes the proteins belonging to the FCP family that were identified in this study in both T. pseudonana and P. tricornutum. They are separated into those originating from the detached FCP bands (bands 5 and 6 in Figure 1A) and FCPs present in the PS I complex (band 1 in Figure 1 and the 20
15 kDa region in Figure 2A,B). Some representatives of the Lhcx clade were only to be detected in the free protein fraction. 5345

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They belong to the PGR-mediated CET around PS I.19,39,40 The PGRL homologue has a weakly conserved PTS but was nevertheless predicted to be chloroplast localized by the protein localization prediction tool HECTAR.33 Figure 5 shows an alignment between the two PGRL homologues from T. pseudonana and P. tricornutum. The PGRL protein in T. pseudonana was predicted to have one transmembrane helix and the P. tricornutum homologue two transmembrane helices.34 For MS sequence coverage of the T. pseudonana PGRL protein; see Supporting Information. 8. Low CO2-Inducible Proteins

Figure 4. Amino acid sequence alignment between two putative low CO2-inducible proteins in T. pseudonana (gi|223999673) and P. tricornutum (gi|219119979). Both were identified on the protein level in the PS II monomer lane as nucleus-encoded putative sequences.

Seven different proteins were identified to predominantly comprise the FCP trimers (band 6 in Figure 1A) in T. pseudonana (Lhcf1/2, 4, 5, 6, 7, 8/9 and one predicted protein). Although encoded by separate genes, the mature Lhcf1 and 2 proteins are identical, which means that they cannot be distinguished by MS analysis. The same applies to Lhcf8/9 and Lhcx1/2 in T. pseudonana, which produce the same set of peptides in MS analysis. The detached antenna was found to be made up exclusively of Lhcf-type proteins. FCP8 (Lhcf8/9) was shown to be the predominant component of higher FCP oligomers in T. pseudonana in two separate thylakoid preparations. In contrast to the main detached antenna, PS I-specific FCPs contained both Lhcr- and Lhcf-type proteins with Lhcr being the predominant type (Table 1). Five different LI818-like proteins were identified in T. pseudonana in general. Figure 1B summarizes their distribution among protein complexes after BN electrophoresis of LL and HL treated samples. Lhcx6_1 and Lhcx4 were detected in the PS I band. Three LI818-like proteins, Lhcx1/2, Lhcx4, and Lhcx6_1, were identified as part of the FCP antennae under HL. Lhcx5 and Lhcx6 were only detected in the free protein fraction of HL treated samples. In P. tricornutum, Lhcx1 was detected as part of PS I, while both Lhcx1 and Lhcx2 were detected in the free protein fraction of LL cultures (see Table 1). 7. Free Protein Fraction

The free protein fraction comprises of all loose proteins of different origin that form a quickly moving band during gel electrophoresis. Most importantly, the free protein fraction in this study contained the five subunits of the diatom OEC (PsbO, PsbQ0 , Psb31, PsbV, and PsbU). It was also abundant in LI818like proteins (two in P. tricornutum and five in T. pseudonana after HL treatment; see also FCP section and Figure 1B). Another significant finding was that PGR5 and PGR5-like (PGRL) homologues were present in the free protein fraction of T. pseudonana.

Two putative low CO2-inducible proteins were identified in the present study. (1) A prominent protein spot was detected between the PS II dimer and PS II monomer lanes in T. pseudonana grown as a batch culture (CO2-limited) but was completely missing in cultures purged with air (CO2-replete, Figure 2A,D). This fact already suggested an involvement of the protein in CO2 acquisition or concentration. This assumption was further strengthened by an N-terminal part sequence homology to limiting CO2-inducible proteins in Chlamydomonas reinhardtii. The matched DB entry is from a protein with an estimated size of 92 kDa (gi|223992735). However, protein sequence coverage was limited to the N-terminus of the DB entry (for sequence coverage see Supporting Information), and the protein’s position in the gel suggested a size of approximately 35 kDa. This would mean that the sequence coverage of 16% given for this protein (Table 1) was severely underestimated. (2) Unlike the above protein, which was only to be identified in samples grown under low CO2, a second possible low CO2-inducible protein (gi|223999673) was detected as part of the PS II monomer band (Table 1) in a number of different samples, including CO2-replete cells under LL as well as cultures treated with HL for up to 8 h. The protein shows homology to the bestrophin superfamily, is present in both diatom species alike (see Figure 4), and again shows homology to low CO2-inducible proteins from C. reinhardtii. It possesses a clear PTS. 9. Comparison between T. pseudonana and P. tricornutum

A comparative 2D BN/SDS-PAGE study of the main photosynthetic complexes in P. tricornutum (Figure 2B) showed protein patterns very similar to the ones shown in detail for T. pseudonana. The detached FCP antenna from P. tricornutum obtained in this study was mainly comprised of nine different proteins, all belonging to the Lhcf type (Lhcf1, 2, 3/4, 5, 8, 9, 10, 11, 12), while in T. pseudonana seven different FCPs were clearly distinguishable (Table 1). PS I-specific FCPs were again a mixture of Lhcr and Lhcf proteins in P. tricornutum. In addition, one early light induced protein (Lhl type) was identified to be associated with PS I. It also belongs to the FCP protein family. In this study, two LI818-like proteins could be detected in the free protein fraction of P. tricornutum cells taken from standard growth conditions. In addition, Lhcx1 was detected as part of the PS I lane (see Figure 2). In T. pseudonana, on the other hand, five different proteins of the LI818 clade were identified in the present study. The P. tricornutum homologue of the putative novel 10 kDa PS I-associated protein found in T. pseudonana was also detected on the protein level, albeit with a low score (data not shown). With a 5346

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Figure 5. Amino acid sequence alignment between two putative PGRL homologues in T. pseudonana (gi|223999427) and P. tricornutum (gi|219110655). Both were identified on the protein level in the free protein fraction as nucleus-encoded putative sequences.

database entry of approximately 42 kDa, it is predicted to be considerably larger than its T. pseudonana counterpart. However, only the N-terminus shows homology to the protein found in T. pseudonana as well as to one predicted protein in the filamentous brown alga Ectocarpus siliculosus. The MS coverage obtained for this protein is too low to be able to make any reliable statement, but the mature protein in P. tricornutum is most likely also shorter than 10 kDa in size. A PGRL homologue as well as one low-CO2-induced bestrophin-like protein could also be identified in P. tricornutum, leading to the conclusion that those features are common to both diatoms.

’ DISCUSSION To our knowledge, the present work is so far the most indepth study of the overall protein composition of diatom thylakoid membranes. Despite the presence of complete genome sequences and gene expression tools, analysis on the protein level is vital for advancing the genome-derived protein models and the elucidation of protein localization and function. Our approach to studying the overall macromolecular organization of diatom photosynthetic complexes was mainly based on BN gel electrophoresis. The pore size of the stacking and separation gels was varied in an attempt to achieve the fullest possible separation of megacomplexes.30 However, the existence of PS II supercomplexes or megacomplexes consisting of both PS I and PS II could not be established. The emerging picture of diatom thylakoid protein complexes and their identified subunits is summarized in Figure 6. This preliminary model is based on the results of the present study as well as the findings of Nagao et al.18 Details on the protein composition of the peripheral light harvesting antenna are summarized in Table 1. 1. Diatom-Specific Features of Photosystem I

After separation of the main photosynthetic complexes by BN gel electrophoresis (Figure 1), PS I appeared as a very broad band but still recognizable as a monomer, confirming earlier reports that PS I is a monomer in diatoms.15,16 This is comparable with

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Figure 6. Schematic overview of the main photosynthetic electron transport complexes in the diatom T. pseudonana. PS II forms dimers, PS I is a monomer. Subunits encircled in solid lines were identified by MS analysis in this study. PS I subunits in dotted lines (PsaE, PsaJ, PsaM, and PsaI) represent short polypeptides detected on the protein level by Nunn et al.16 The position of peripheral PS I subunits in the scheme is random, since there is no experimental data available. X: putative novel PS I protein (gi|223993351), XX: putative T. pseudonana-specific protein (gi|223995405), XXX: HL-induced (represented by dashed line) putative lumen protein (gi|224013736) associated with PS I, PQ: plastoquinone, Cyt: cytochrome. A putative NDH-2 is predicted to be plastid localized and assist the PS I-mediated CET in T. pseudonana. The oxygen evolving complex of PS II consists of PsbO (O), PsbU (U), PsbV (V), Psb31, PsbQ0 (Q0 ), according to Nagao et al.10 and Table 1. For a detailed distribution of FCPs, see Table 1; for a distribution of Lhcx proteins, see Figure 1B.

the behavior of Arabidopsis PS I in BN gels41 and unlike cyanobacteria,42 where the PsaL subunit is responsible for PS I trimer formation43 (reviewed in Grotjohann and Fromme44). However, some degree of differentiation in PS I complexes was clearly visible as PS I appeared as two to three bands in close proximity to each other. As revealed by 2D BN/SDS-PAGE, this subdivision of the PS I band was due to different amounts, maybe even a different composition, of FCP bound to the respective PS I complex (Figure 2). In the present study, three additional PS I subunits were identified on the protein level compared to an earlier study on diatom PS I particles:16 PsaF, PsaC, PsaD. Apart from those plastid-encoded proteins known to belong to PS I, two further nucleus-encoded proteins are likely to be associated with PS I. The present study raises the possibility that despite a high level of conservation between photosynthetic organisms, the diatom PS I contains subunits that are unique to this organism group. The two novel plastid-targeted proteins were identified earlier in a proteomics study on whole T. pseudonana cells.24 Here, they were shown to be associated with PS I after detergent treatment, suggesting them to be genuine PS I subunits. In the diatom Chaetoceros gracilis, one extrinsic PS II protein (Psb31) was identified and later described to be unique to the OEC of diatoms,18,35,45 so our finding on PS I is consistent with diatom-specific subunits being present in photosynthetic complexes. The first new diatom-specific PSI protein (gi|223993351) is a subunit below 10 kDa in size and is found in both diatoms studied here. This protein was clearly visible as a separate spot and was found in all PS I complexes in the 2D BN/SDS-PAGE gels tested in this study. So far, no prediction on a putative function could be given based on sequence homology because this protein is diatom-specific. The second one (gi|223995405) was found to be around 20 kDa in size and has no counterparts in other organisms. Having no homologue in P. tricornutum either, this protein appears to be unique to T. pseudonana, suggesting a function specific to centric diatoms. This latter protein displays a 5347

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Journal of Proteome Research very weak similarity to bacterial NAD(P)H dehydrogenases (NDH). Both of the above proteins display no transmembrane helix sequences34 and are therefore shown as peripheral subunits in Figure 6. A third novel protein (gi|224013736) was only detected in association with PS I in HL-treated samples, implicating a role in PS I stability. Its PTS is poorly conserved (AF), but the protein was nevertheless predicted to be chloroplastlocalized by HECTAR.33 It also possesses the double arginine and thylakoidal processing peptidase motifs, suggesting it to be lumen localized. This putative protein has a P. tricornutum homologue and shows sequence similarity to lumen proteins of unknown function in other photosynthetic organisms. It is evident that the elucidation of the function of the novel diatom-specific PS I subunits discovered here requires much more extensive research and different methodological approaches. Compared to PS I in Arabidopsis (reviewed by Jensen et al.46), diatoms lack close homologues of the subunits PsaG, H, N, and O. PsaH and PsaO are suggested to be involved in state transitions in plants. Considering the fact that no indications of state transition have ever been found in diatoms,13 this appears only logical. The subunits F and N form the likely docking site for plastocyanin. Diatoms only possess PsaF. However, plastocyanin is replaced by cytochrome c6 in this organism group,47 which might account for the difference. Diatoms also possess the subunits PsaI (SU 8) and J (SU 9), but they are more closely related to other algal groups than to plants. 2. PS I-Mediated Cyclic Electron Transport Has SpeciesSpecific Features in Diatoms

One specific goal of this study was to find proteins that might be involved in CET around PS I, which in biophysical experiments was previously shown to have high activity in diatoms.26 For this purpose, three mechanisms known from other organisms were taken into account: (1) a multisubunit NDH complex, (2) single-subunit type II NDH, and (3) PGR-like proteins. (1) Unlike higher plants, the chloroplast genome of diatoms does not encode any NDH subunits. This fact led to the conclusion that a multisubunit NDH-like complex is either missing in diatoms or the responsible genes are nucleus-encoded. A homology-based search for putative NDH subunits in T. pseudonana revealed only two proteins with weak similarity to the NdhK and NdhI subunits of the plastid NDH complex in Synechocystis sp. PCC 680321,48 and Arabidopsis.49 However, the diatom NdhI homologue did not show any apparent PTS, and the NdhK homologue displayed a stronger sequence homology to a putative mitochondrial complex I subunit. Thus, there is compelling evidence on the genetic level that a plastid multisubunit NDH complex is not present in diatoms. On the protein level, any search for plastidtargeted NDH subunits under standard growth conditions has been unsuccessful. In conclusion, the existence of a multisubunit NDH complex in diatoms appears highly unlikely based on the data presented here. (2) On the genetic level, we were able to indentify one T. pseudonana-specific plastid-targeted protein that has sequence similarity to type II dehydrogenases from C. reinhardtii and Synechocystis species. In T. pseudonana, EST counts studies50 indicate that this protein is only expressed under certain conditions, for example, silica limitation. So far, we were unable to identify this NDH on the protein level. This is not surprising, since this protein

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is predicted to be soluble and therefore not an integral part of the thylakoid membrane. An interesting fact is that this type II NDH from T. pseudonana lacks a close plastidtargeted homologue in the genome of P. tricornutum. Earlier physiological evidence in the centric diatom C. meneghiniana suggested that a single-subunit type II NDH might be involved in CET around PS I.26 It was shown that under PS II inhibition, PS I could be reduced by alternative electrons in C. meneghiniana and no NADPH limitation was indicated. This process did not lead to the generation of a significant proton gradient, however. This was in contrast to P. tricornutum, where under PS II inhibition a proton gradient strong enough to induce nonphotochemical quenching (NPQ) was generated. However, PS II inhibition seemed to lead to NADPH limitation in P. tricornutum.26 For these reasons, it was both surprising and significant to find no indication of a multisubunit NDH-like protein complex in P. tricornutum neither on the genetic nor the protein level in the present study. (3) The most significant finding related to CET around PS I was the identification of PGR5 and PGRL homologues in the free protein fractions of T. pseudonana and P. tricornutum thylakoid membranes. PGR/PGRL proteins mediate a form of CET that transfers electrons from ferredoxin to the plastoquinone (PQ) pool/Cyt b6/f complex.39,51 The PGRL target sequences in both diatoms only displayed the highly conserved AF motif (see Figure 5). HECTAR33 nevertheless predicted them to be plastid targeted. The PGR5 homologue in T. pseudonana, on the other hand, was not. However, its amino acid sequence contains the motif GFAP. It has been shown that A at the
1 position can be substituted for G, still enabling chloroplast protein import in diatoms.52 A protein secondary structure prediction tool34 suggested that the diatom PGRL protein forms one transmembrane helix in T. pseudonana and two helices in P. tricornutum. This would be comparable to the PGRL1 protein in Arabidopsis which is known to have a transmembrane domain51 as opposed to PGR5 which is a soluble protein. In plants, the two proteins (PGR5 and PGRL1) are known to form complexes and mediate a switch between linear and cyclic electron transport.51 The findings presented here provide proof that components of PGR-mediated CET around PS I are present in diatoms. This pathway was shown to complement photosynthesis in higher plants to a significant degree19 and the extent of its contribution to diatom photosynthesis, as well as the existence of PGR complexes, remain to be established. On the basis of the present study, the PS I CET appears to be mainly PGR-mediated throughout diatom species and additionally assisted by type II NDH in centric diatoms. Type II NDHmediated electron transfer would result in an electron flow from ferredoxin to the plastoquinone pool (PQ)/cytochrome b6/f complex. The difference in NADPH availability between P. tricornutum and centric diatoms could in this case still be due to the activity of a putative plastid terminal oxidase (PTOX) oxidizing the PQ pool. Homologues of the Arabidopsis IMMUTANS protein, acting as a plastid PQ-dependent oxidase,53,54 are present in both diatoms studied here. In T. pseudonana, the respective protein displays a well conserved PTS. In P. tricornutum, two putative IMMUTANS homologues were discovered on the genetic level, but their target sequences are not clearly defined. 5348

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Journal of Proteome Research As is the case with mitochondrial and plastid NDH subunits, caution is necessary when designating a putative protein as PTOX because of its possible similarity with mitochondrial alternative oxidases. Type II dehydrogenases are known to participate in CET in C. reinhardtii.55 In view of the findings of the present study, centric diatoms appear to perform a PS I CET of the same type as the green alga C. reinhardtii. CET around PS I can occur under conditions of PS II limitation or be implemented in CCM in cyanobacteria (reviewed by Battchikova et al.56). When operating alongside the linear electron transport, CET is essential for the adjustment of NADPH/ATP ratios because it enhances the proton gradient across the thylakoid membrane, leading to ATP generation without net NADPH production. Therefore, the protein composition of cultures under different physiological conditions will still have to be extensively studied before the precise mechanism and the contribution of different proteins toward PS I-mediated CET can be finally elucidated. 3. There Are No PS II Supercomplexes in Diatoms

The PS II protein composition of T. pseudonana found in this study confirmed previous studies on PS II particles from the diatom C. gracilis18,35 as well as previous suggestions that in diatoms PS II forms dimers in vivo. The diatom species studied here were not found to form any PS II supercomplexes as observed in higher plants,41 despite applying the novel large pore BN-gels as described in Sirpi€o et al.30 4. Putative Diatom Low CO2-Inducible Proteins Involved in Ci Acquisition

Low CO2-inducible proteins are of particular interest in connection with aquatic photosynthetic organisms because of the wide distribution of CCM among them. Most detailed knowledge of CCM is still based on studies with cyanobacteria (reviewed by Price et al.57), but C. reinhardtii also plays an important role as a model organism for eukaryotes (reviewed by Grossman et al.58). A clear-cut case of a low CO2-inducible protein complex bigger than 480 kDa could be shown for T. pseudonana. This size estimation is based upon previous data suggesting a size of 440
480 kDa for PS II in diatoms.59 However, only one subunit at approximately 35 kDa (gi|223992735) could be identified in this lane (Figure 2A), so this protein possibly forms a high oligomeric structure. The DB entry for this protein is considerably larger in size than the 35 kDa protein observed in this study. The discrepancy between predicted and actual protein size in the gel pointed to an incorrect DB annotation. The most likely explanation is that the actual protein consists of only one exon (as opposed to two estimated in the DB), which would result in a mature protein of approximately 35 kDa. It is also worth mentioning that the first part of the DB sequence is that of a soluble protein, while the second one is predicted to form transmembrane helices. A putative function is suggested both by N-terminal C. reinhardtii homology and the observation of a long-term CO2-level dependent expression of the protein. Another chloroplast-targeted low CO 2 -inducible protein (gi|223999673 in T. pseudonana and gi|219119979 in P. tricornutum) was identified as part of the PS II monomer band in both diatoms studied here. This apparent PS II association, however, can also be due to an artifact of the thylakoid isolation or separation protocol. The protein belongs to the bestrophin protein family. Members of this family are believed to be chloride (Cl
)

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channels60 and to be membrane-associated. The two proteins found in this study bear close similarity to proteins from green algae, mosses, and plants. They are predicted to form two transmembrane spanning helices,34 which is also in accordance with other known bestrophin family members. 5. Novel Insights into FCP Composition and Distribution of LI818-like Proteins in Diatom Thylakoids

Light harvesting antennae represent a very prominent part of the overall protein content of the thylakoid membrane and were in fact shown to be the most highly expressed proteins in wholecell lysates of T. pseudonana.24 An allocation of the main FCP antennae to PS II in diatoms is experimentally challenging because FCPs detach easily from PS II during detergent treatment. So far, PS II particles retaining attached FCPs have been isolated from the diatom C. gracilis.35 However, no extensive characterization of their protein composition was carried out. More recent studies also showed the existence of integral PS I-specific FCPs,15 which mediate excitation energy between the more loosely attached peripheral antennae and the PS I core.16 An extensive MS-based study of the precise protein composition of detached, oligomeric FCPs and PS I-associated FCPs in P. tricornutum was published only recently.17 The present study advances the knowledge on the protein composition of the PS I-specific and peripheral antennae of the centric diatom species T. pseudonana. Three main groups of light harvesting proteins can be distinguished in diatoms: Lhcf, Lhcr and Lhcx. No affiliation of FCPs to PS II could be observed under the thylakoid solubilization treatment used in this study. The peripheral antenna, believed to be common to both PS II and PS I, was shown to be exclusively comprised of Lhcf proteins in this study. In T. pseudonana, the main FCP fractions isolated were most probably trimers, based on former studies14,17 as well as their clear separation from the free protein fraction in BN electrophoresis (Figure 1A). A higher FCP oligomer obtained at lower detergent concentrations was possibly composed of a multifold of trimers and was shown to be predominantly made up of Lhcf8/9. Contrary to PS II, specific FCPs remained attached to PS I after detergent solubilization. The PS I-specific core antenna of T. pseudonana was found to be mainly comprised of Lhcr proteins. In addition, two further members of the FCP protein family were detected, which however are still designated as “predicted proteins” in the DB. Lhcx genes encode a third important subspecies of FCPrelated proteins in diatoms. They have been shown to participate in the generation of NPQ and the response to HL stress61,62 and show homology to LI818 proteins in C. reinhardtii.63,64 Diatoms lack a close PsbS homologue, but it has been suggested that Lhcx proteins might have a similar function instead. Five Lhcx proteins were found in T. pseudonana in the present study: Lhcx1/2, 4, 5, 6, 6_1. Figure 1B shows the distribution of Lhcx proteins among the complexes separated by BN electrophoresis. Other studies have already shown that Lhcx1 is expressed under LL conditions in P. tricornutum,61 and both Lhcx1 and Lhcx2 were detected on the protein level.17 In cultures taken from LL growth conditions, three Lhcx proteins were identified (Lhcx6_1, Lhcx1/2, and Lhcx5). One of them (encoded by Lhcx6_1) was identified as part of PS I as well as of the detached FCP timers and the free protein fraction. This is an intriguing finding and suggests that Lhcx6_1 might be involved in PS I-specific photoprotection or have an unknown 5349

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Journal of Proteome Research function. Under HL treatment, Lhcx4 could be detected as part of PS I in addition to the Lhcx6_1 gene product. Lhcx1/2 was found as part of FCP timers under HL and in association with PS I in silica-limited cells, suggesting a general stress responserelated function of Lhcx proteins extending beyond NPQ formation. Lhcx5 and Lhcx6 were only detected in the free protein fraction, so no statement can be made about their localization at present. It has already been shown at the protein level that Lhcx1 is present in considerable amounts under LL and is upregulated by HL treatment, while Lhcx6 is upregulated at the beginning of a HL phase in T. pseudonana.62 One Lhcx-type protein was earlier suggested to be a component of the trimeric FCPa in another centric diatom, C. meneghiniana.65 The expression of Lhcx6_1, which was identified both in the FCP fraction and in association with PS I in the present study, has not been reported previously. It has to be pointed out that the products of Lhcx1 and Lhcx2 could not be distinguished on the protein level by MS analysis. This was because the mature proteins differ in a single amino acid position (V instead of I) which, however, was not included in the peptide coverage obtained here. Richard et al.64 showed that the C. reinhardtii LI818 protein is not tightly embedded in the thylakoid membrane and is most likely stroma exposed. In accordance with this observation and unlike Lhcf and Lhcr proteins, the Lhcx proteins identified in the present study were mostly predicted to be soluble. The only exception was Lhcx5 which was predicted to have one transmembrane helix.34 Earlier gene expression studies in P. tricornutum61 suggested that under LL mainly one protein (Lhcx1) from the LI818 clade is present. We were able to detect both Lhcx1 and Lhcx2 in LLtreated P. tricornutum cultures with a high score. Still, the MS analysis carried out in the present study is by no means quantitative, so no conclusions can be drawn on the abundance of each Lhcx protein. Very likely, Lhcx1 is still the predominant protein under low illumination. The results presented here are the most detailed protein data on Lhcx protein expression and localization in diatom thylakoids so far. 6. Lumen Localized Proteins

The free protein fraction contained a number of proteins that were either detached from multisubunit protein complexes during thylakoid isolation/detergent treatment or represented single subunits that were not associated with any protein complex. In addition, lumenal proteins with a peripheral thylakoid membrane association were also found in the free protein fraction in this study. One protein predicted to be lumen localized and found in this study was cytochrome c6. This fact is worth mentioning because diatoms are known to have cytochrome c6 operating as an electron carrier between the Cytb6/f complex and PS I instead of plastocyanin.47 7. Diversity of Plastid Target Sequences in Diatoms

In this study, two approaches for protein plastid localization prediction were used. First, putative plastid target sequences were deduced manually, based on the studies of Kilian and Kroth66 and Gruber et al.,52 and second, the heterokont-specialized online tool for plastid target sequence prediction HECTAR33 was used. Roughly, ASAF- or AFAP-motifs found at the beginning of precursor protein sequences were considered sufficient evidence to predict chloroplast localization. As is known from deletion mutations, the phenylalanine at the +1 position (first amino acid after cleavage site) is highly conserved but can be substituted by tryptophan in some

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cases.52,66 Commonly available online tools for plastid target prediction (targetP and chloroP67) were found to be unsuitable for diatoms when tested with known plastid proteins. This is why HECTAR, which was specifically designed for heterokonts, was employed.33 Using this tool, several proteins were correctly predicted to be chloroplast localized despite some clear divergences from the conserved ASAFAP motif. Those included the two diatom PGRL proteins and the P. tricornutum homologue of the PGR5 protein identified here. The latter could not be predicted to be chloroplast targeted by HECTAR, although it displays the domain GFAP. An extensive study on plastid target sequences in diatoms has shown that alanine at the
1 position can be substituted for glycine, still enabling chloroplast protein import in diatoms.52 On the basis of this fact and the suggested function of the protein, plastid localization appears highly likely. Some of the nucleus-encoded, plastid-targeted proteins identified in T. pseudonana notably had close homologues in P. tricornutum displaying PTS diverging from the known pattern. One instance was a sequence alignment between the putative low-CO2 induced protein found in this study (gi|223992735) and its closest P. tricornutum homologue. The plastid target sequence in the pennate diatom only displayed the conserved AF motif. HECTAR33 still confirmed both proteins to be plastid targeted. 8. Concluding Remarks and Outlook

The present study raises the exciting possibility of diatomspecific features in the very architecture of PS I and paves the way for a better structural and evolutionary understanding of diatom photosynthetic complexes. It will hopefully also provide a broad basis for further research into the mechanism of PS I-mediated CET. One of the main tasks ahead is the establishing of a physiological function for the putative photosynthetic proteins discovered so far. It is a challenging task since many of them seem to be unique to diatoms or only slightly related to any proteins of known function. But it also has to be pointed out that when speaking of T. pseudonana-specific proteins in this study, it is highly likely that they are common to other centric diatoms as well. It is probably the lack of genomic information in the DB that makes proteins appear unique to T. pseudonana at this stage. Obtained proteome data suggests important candidates for future mutagenesis analysis in diatoms. To this end, the generation of T. pseudonana RNAi strains of (1) novel putative PS I proteins, (2) one putative type II NDH, and (3) one putative PGRL protein will be a major priority. Mutants to be subsequently tested for linear and cyclic electron transport capacities under PS II inhibition as well as carbon limitation will provide valuable information about dynamics and regulation of diatom photosynthesis.

’ ASSOCIATED CONTENT

bS

Supporting Information MS sequence coverage of novel predicted proteins, Mascotbased peptide view for single peptide protein identification. This material is available free of charge via the Internet at http://pubs. acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Telephone: +358 2 333 8071. Fax: +358 2 333 8075. E-mail: evaaro@utu.fi. 5350

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’ ACKNOWLEDGMENT This work was supported by the Marie Curie Initial Training Networks COSI (GA-215174) and HARVEST (PITN-GA2009-238017), the Academy of Finland (CoE project 118637), and the EU FP7/Energy Network project SOLAR-H2 (contract 212508). MS analyses were performed at the Proteomics core facility of Turku Centre for Biotechnology. The expert technical assistance of Arttu Heinonen is gratefully acknowledged. ’ ABBREVIATIONS CCM: carbon concentrating mechanisms; CET: cyclic electron transport; Cyt: cytochrome; DB: database; DM: n-dodecyl-β-Dmalatoside; FCP: fucoxanthin-chlorophyll a/c-binding proteins; HL: high light; LC: liquid chromatography; LL: low light; MS: mass spectrometry; NDH: NAD(P)H dehydrogenase; OEC: oxygen evolving complex; PGR: proton gradient regulation; PGRL: PGR5-like; PS: photosystem; PTS: plastid (chloroplast) target signal ’ REFERENCES (1) Behrenfeld, M. J.; Falkowski, P. G. Photosynthetic rates derived from satellite-based chlorophyll concentration. Limnol. Oceanogr. 1997, 42 (1), 1–20. (2) Falkowski, P. G. The role of phytoplankton photosynthesis in global biogeochemical cycles. Photosynth. Res. 1994, 39, 235–258. (3) Falkowski, P. G.; Barber, R. T.; Smetacek, V. Biogeochemical controls and feedbacks on ocean primary production. Science 1998, 281, 200–206. (4) Bhattacharya, D.; Archibald, J. M.; Weber, A. P. M.; Reyes-Prieto, A. How do endosymbionts become organelles? Understanding early events in plastid evolution. BioEssays 2007, 29, 1239–1246. (5) Pyszniak, A. M.; Gibbs, S. P. Immunocytochemical localization of photosystem I and the fucoxanthin-chlorophyll a/c light-harvesting complex in the diatom Phaeodactylum tricornutum. Protoplasma 1992, 166, 208–217. (6) Berkaloff, C.; Duval, J. C.; Hauswirth, N.; Rousseau, B. Freeze fracture study of thylakoids of Fucus serratus. J. Phycol. 1983, 19, 96–100. (7) Andersson, B.; Anderson, J. M. Lateral heterogeneity in the distribution of chlorophyll-protein complexes of the thylakoid membranes of spinach chloroplasts. Biochim. Biophys. Acta 1980, 593, 427–440. (8) Tikkanen, M.; Grieco, M.; Aro, E.-M. Novel insights into light harvesting complex II phosphorylation and “state transitions. Trends Plant Sci. 2011, 16 (3), 126–131. (9) Tikkanen, M.; Grieco, M.; Kangasj€arvi, S.; Aro, E.-M. Thylakoid protein phosphorylation in higher plant chloroplasts optimizes electron transfer under fluctuating light. Plant Physiol. 2010, 152, 723–735. (10) Bennett, J. Phosphorylation of chloroplast membrane polypeptides. Nature 1977, 269, 344–346. (11) Bennett, J. Regulation of photosynthesis by reversible phosphorylation of the light harvesting chlorophyll a/b protein. Biochem. J. 1983, 212, 1–13. (12) Murata, N. Control of excitation transfer in photosynthesis. I. Light-induced change in chlorophyll a fluorescence in Porphyridium cruentum. Biochim. Biophys. Acta 1969, 172, 242–251. (13) Owens, T. G. Light-harvesting function in the diatom Phaeodactylum tricornutum: II. Distribution of excitation energy between the photosystems. Plant Physiol. 1986, 80, 739–746. (14) B€uchel, C. Fucoxanthin-chlorophyll proteins in diatoms: 18 and 19 kDa subunits assemble into different oligomeric states. Biochemistry 2003, 42, 13027–13034. (15) Veith, T.; B€uchel, C. The monomeric photosystem I-complex of the diatom Phaeodactylum tricornutum binds specific fucoxanthin

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chlorophyll proteins (FCPs) as light-harvesting complexes. Biochim. Biophys. Acta 2007, 1767, 1428–1435. (16) Ikeda, Y.; Komura, M.; Watanabe, M.; Minami, C.; Koike, H.; Itoh, S.; Kashino, Y.; Satoh, K. Photosystem I complexes associated with fucoxanthin-chlorophyll-binding proteins from a marine centric diatom; Chaetoceros gracilis. Biochim. Biophys. Acta 2008, 1777, 351–361. (17) Lepetit, B.; Volke, D.; Gilbert, M.; Wilhelm, C.; Goss, R. Evidence for the existence of one antenna-associated, lipid-dissolved, and two protein-bound pools of diadinoxanthin cycle pigments in diatoms. Plant Physiol. 2010, 154 (4), 1905–1920. (18) Nagao, R.; Moriguchi, A.; Tomo, T.; Niikura, A.; Nakajima, S.; Suzuki, T.; Okumura, A.; Iwai, M.; Shen, J.-R.; Ikeuchi, M.; Enami, I. Binding and functional properties of five extrinsic proteins in oxygenevolving photosystem II from a marine centric diatom; Chaetoceros gracilis. J. Biol. Chem. 2010, 285 (38), 29191–29199. (19) Munekage, Y.; Hashimoto, M.; Miyake, C.; Tomizawa, K.-I.; Endo, T.; Tasaka, M.; Shikanai, T. Cyclic electron flow around photosystem I is essential for photosynthesis. Nature 2004, 429, 579–582. (20) Zhang, P.; Battchikova, N.; Jansen, T.; Appel, J.; Ogawa, T.; Aro, E.-M. Expression and functional roles of the two distinct NDH-1 complexes and the carbon acquisition complex NdhD3/NdhF3/CupA/ Sll1735 in Synechocystis sp PCC 6803. Plant Cell 2004, 16, 3326–3340. (21) Battchikova, N.; Aro, E.-M. Cyanobacterial NDH-1 complexes: multiplicity in function and subunit composition. Physiol. Plant 2007, 131, 22–32. (22) Armbrust, E. V.; Berges, J. A.; Bowler, C.; Green, B. R.; Martinez, D.; Putnam, N. H.; Zhou, S.; Allen, A. E.; Apt, K. E.; Bechner, M.; Brzezinski, M. A.; Chaal, B. K.; Chiovitti, A.; Davis, A. K.; Demarest, M. S.; Detter, C. J.; Glavina, T.; Goodstein, D.; Hadi, M. Z.; Hellsten, U.; Hildebrand, M.; Jenkins, B. D.; Jurka, J.; Kapitonov, V. V.; Kr€oger, N.; Lau, W. W. Y.; Lane, T. W.; Larimer, F. W.; Lippmeier, J. C.; Lucas, S.; Medina, M.; Montsant, A.; Obornik, M.; Schnitzler-Parker, M.; Palenik, B.; Pazour, G. J.; Richardson, P. M.; Rynearson, T. A.; Saito, M. A.; Schwartz, D. C.; Thamatrakoln, K.; Valentin, K.; Vardi, A.; Wilkerson, F. P.; Rokhsar, D. S. The genome of the diatom Thalassiosira pseudonana: ecology; evolution and metabolism. Science 2004, 306, 79–86. (23) Bowler, C.; Allen, A. E.; Badger, J. H.; Grimwood, J.; Jabbari, K.; Kuo, A.; Maheswari, U.; Martens, C.; Maumus, F.; Otillar, R. P.; Rayko, E.; Salamov, A.; Vandepoele, K.; Beszteri, B.; Gruber, A.; Heijde, M.; Katinka, M.; Mock, T.; Valentin, K.; Verret, F.; Berges, J. A.; Brownlee, C.; Cadoret, J.-P.; Chiovitti, A.; Choi, C. J.; Coesel, S.; De Martino, A.; Detter, J. C.; Durkin, C.; Falciatore, A.; Fournet, J.; Haruta, M.; Huysman, M. J. J.; Jenkins, B. D.; Jiroutova, K.; Jorgensen, R. E.; Joubert, J.; Kaplan, A.; Kr€oger, N.; Kroth, P. G.; La Roche, J.; Lindquist, E.; Lommer, M.; Martin-Jezequel, V.; Lopez, P. J.; Lucas, S.; Mangogna, M.; McGinnis, K.; Medlin, L. K.; Montsant, A.; Oudot-Le Secq, M.-P.; Napoli, C.; Obornik, M.; Schnitzler Parker, M.; Petit, J.-L.; Porcel, B. M.; Poulsen, N.; Robison, M.; Rychlewski, L.; Rynearson, T. A.; Schmutz, J.; Shapiro, H.; Siaut, M.; Stanley, M.; Sussman, M. R.; Taylor, A. R.; Vardi, A.; von Dassow, P.; Vyverman, W.; Willis, A.; Wyrwicz, L. S.; Rokhsar, D. S.; Weissenbach, J.; Armbrust, E. A.; Green, B. R.; Van de Peer, Y.; Grigoriev, I. V. The Phaeodactylum genome reveals the evolutionary history of diatom genomes. Nature 2008, 456, 239–244. (24) Nunn, B. L.; Aker, J. R.; Shaffer, S. A.; Tsai, Y.; Strzepek, R. F.; Boyd, P. W.; Freeman, T. L.; Brittnacher, M.; Malmstr€om, L.; Goodlett, D. R. Deciphering diatom biochemical pathways via whole-cell proteomics. Aquat. Microb. Ecol. 2009, 55 (3), 241–253. (25) Carvalho, R. N.; Lettieri, T. Proteomic analysis of the marine diatom Thalassiosira pseudonana upon exposure to benzo(a)pyrene. BMC Genomics 2011, 12, 159. (26) Grouneva, I.; Jakob, T.; Wilhelm, C.; Goss, R. The regulation of xanthophyll cycle activity and of non-photochemical fluorescence quenching by two alternative electron flows in the diatoms Phaeodactylum tricornutum and Cyclotella meneghiniana. Biochim. Biophys. Acta 2009, 1787, 929–938. (27) Guillard, R. R. L.; Ryther, J. H. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea Cleve. Can. J. Microbiol. 1962, 1962 (8), 229–239. 5351

dx.doi.org/10.1021/pr200600f |J. Proteome Res. 2011, 10, 5338–5353

Journal of Proteome Research (28) Guillard, R. R. L. Culture of phytoplankton for feeding marine invertebrates. In Culture of Marine Invertebrate Animals; Smith, W. L.; Chanley, M. H., Eds.; Plenum Press: New York, 1975; pp 26
60. (29) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the folin phenol reagent. J. Biol. Chem. 1951, 193, 265–275. (30) Sirpi€o, S.; Suorsa, M.; Paakkarinen, V.; Aro, E.-M. Optimized native gel systems for separation of thylakoid protein complexes: novel super- and megacomplexes. Biochem. J. 2011, Immediate Publication, doi:10.1042/BJ20102155. (31) Blum, H.; Beier, H.; Gross, H. J. Improved silver staining of plant proteins; RNA and DNA in polyacrylamide gels. Electrophoresis 1987, 8, 93–99. (32) Shevchenko, A.; Tomas, H.; Havlis, J.; Olsen, J. V.; Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 2007, 1 (6), 2856–2860. (33) Gschloessl, B.; Guermeur, Y.; Cock, J. M. HECTAR: A method to predict subcellular targeting in heterokonts. BMC Bioinformatics [Online] 2008, 9 (393). http://www.biomedcentral.com/1471-2105/ 9/393 (accessed June 10, 2011). (34) Hirokawa, T.; Boon-Chieng, S.; Mitaku, S. SOSUI: classification and secondary structure prediction system for membrane proteins. Bioinformatics Applic. Note 1998, 14 (4), 378–379. (35) Nagao, R.; Ishii, A.; Tada, O.; Suzuki, T.; Dohmae, N.; Okumura, A.; Iwai, M.; Takahashi, T.; Kashino, Y.; Enami, I. Isolation and characterization of oxygen-evolving thylakoid membranes and Photosystem II particles from a marine diatom Chaetoceros gracilis. Biochim. Biophys. Acta 2007, 1767, 1353–1362. (36) Mori, H.; Cline, K. Post-translational protein translocation into thylakoids by the Sec and ΔpH-dependent pathways. Biochim. Biophys. Acta 2001, 1541, 80–90. (37) Boekema, E. J.; B€ottcher, B. The structure of ATP synthase from chloroplasts. Conformational changes of CF1 studied by electron microscopy. Biochim. Biophys. Acta 1992, 1098, 131–143. (38) Groth, G.; Pohl, E. The structure of the chloroplast F1-ATPase at 3.2 Å resolution. J. Biol. Chem. 2001, 276, 1345–1352. (39) Rumeau, D.; Peltier, G.; Cournac, L. Chlororespiration and cyclic electron flow around PSI during photosynthesis and plant stress response. Plant Cell Environ. 2007, 30, 1041–1051. (40) Long, T. A.; Okegawa, Y.; Shikanai, T.; Schmidt, G. W.; Covert, S. F. Conserved role of proton gradient regulation 5 in the regulation of PSI cyclic electron transport. Planta 2008, 228, 907–918. (41) Aro, E.-M.; Suorsa, M.; Rokka, A.; Allahverdiyeva, Y.; Paakkarinen, V.; Saleem, A.; Battchikova, N.; Rintam€aki, E. Dynamics of photosystem II: a proteomic approach to thylakoid protein complexes. J. Exp. Bot. 2004, 56 (411), 347–356. (42) Herranen, M.; Battchikova, N.; Zhang, P.; Graf, A.; Sirpi€o, S.; Paakkarinen, V.; Aro, E.-M. Towards functional proteomics of membrane protein complexes in Synechocystis sp. PCC 6803. Plant Physiol. 2004, 134, 470–481. (43) Chitnis, V. P.; Chitnis, P. R. PsaL subunit is required for the formation of photosystem I trimers in the cyanobacterium Synechocystis sp. PCC 6803. FEBS 1993, 336 (2), 330–334. (44) Grotjohann, I.; Fromme, P. Structure of cyanobacterial Photosystem I. Photosynth. Res. 2005, 85, 51–72. (45) Okumura, A.; Nagao, R.; Suzuki, T.; Yamagoe, S.; Iwai, M.; Nakazato, K.; Enami, I. A novel protein in Photosystem II of a diatom Chaetoceros gracilis is one of the extrinsic proteins located on lumenal side and directly associates with PSII core components. Biochim. Biophys. Acta 2008, 1777, 1545–1551. (46) Jensen, P. E.; Bassi, R.; Boekema, E. J.; Dekker, J. P.; Jansson, S.; Leister, D.; Robinson, C.; Scheller, H. V. Structure, function and regulation of plant photosystem I. Biochim. Biophys. Acta 2007, 1767, 335–352. (47) Inda, L. A.; Erdner, D. L.; Peleato, M. L.; Anderson, D. M. Cytochrome c6 isolated from the marine diatom Thalassiosira weissflogii. Phytochemistry 1999, 51, 1–4. (48) Battchikova, N.; Zhang, P.; Rudd, S.; Ogawa, T.; Aro, E.-M. Identification of NdhL and Ssl1690 (NdhO) in NDH-1L and NDH-1M

ARTICLE

Complexes of Synechocystis sp. PCC 6803. J. Biol. Chem. 2005, 280, 2587–2595. (49) Peng, L.; Yamamoto, H.; Shikanai, T. Structure and biogenesis of the chloroplast NAD(P)H dehydrogenase complex. Biochim. Biophys. Acta 2011, 1807, 945–953. (50) Maheswari, U.; Mock, T.; Armbrust, E. V.; Bowler, C. Update of the Diatom EST Database: a new tool for digital transcriptomics. Nucleic Acids Res. 2009, 37, D1001–D1005. (51) DalCorso, G.; Pesaresi, P.; Masiero, S.; Aseeva, E.; Sch€unemann, D.; Finazzi, G.; Joliot, P.; Barbato, R.; Leister, D. A Complex containing PGRL1 and PGR5 is involved in the switch between linear and cyclic electron flow in Arabidopsis. Cell 2008, 132, 273–285. (52) Gruber, A.; Vugrinec, S.; Hempel, F.; Gould, S. B.; Maier, U.-G.; Kroth, P. G. Protein targeting into complex diatom plastids: functional characterisation of a specific targeting motif. Plant Mol. Biol. 2007, 64, 519–530. (53) Wu, D.; Wright, D. A.; Wetzel, C.; Voytas, D. F.; Rodermel, S. The IMMUTANS variegation locus of Arabidopsis defines a mitochondrial alternative oxidase homolog that functions during early chloroplast biogenesis. Plant Cell 1999, 1999 (11), 43–55. (54) Carol, P.; Stevenson, D.; Bisanz, C.; Breitenbach, J.; Sandmann, G.; Mache, R.; Coupland, G.; Kuntz, M. Mutations in the Arabidopsis gene IMMUTANS cause a variegated phenotype by inactivating a chloroplast terminal oxidase associated with phytoene desaturation. Plant Cell 1999, 11, 57–68. (55) Jans, F.; Mignolet, E.; Houyoux, P.-A.; Cardol, P.; Ghysels, B.; Cuine, S.; Cournac, L.; Peltier, G.; Remacle, C.; Franck, F. A type II NAD(P)H dehydrogenase mediates light-independent plastoquinone reduction in the chloroplast of Chlamydomonas. Proc. Natl. Acad. Sci. U. S. A. 2008, 105 (51), 20546–20551. (56) Battchikova, N.; Eisenhut, M.; Aro, E.-M. Cyanobacterial NDH-1 complexes: Novel insights and remaining puzzles. Biochim. Biophys. Acta 2011, 1807, 935–944. (57) Price, G. D.; Badger, M. R.; Woodger, F. J.; Long, B. M. Advances in understanding the cyanobacterial CO2-concentratingmechanism (CCM): functional components, Ci transporters, diversity, genetic regulation and prospects for engineering into plants. J. Exp. Bot. 2008, 59 (7), 1441–1461. (58) Grossman, A. R.; Croft, M.; Gladyshev, V. N.; Merchant, S. S.; Posewitz, M. C.; Prochnik, S.; Spalding, M. H. Novel metabolism in Chlamydomonas through the lens of genomics. Curr. Opin. Plant Biol. 2007, 10, 190–198. (59) Lepetit, B.; Volke, D.; Szab o, M.; Hoffmann, R.; Garab, G.; Wilhelm, C.; Goss, R. Spectroscopic and molecular characterisation of the oligomeric antenna of the diatom Phaeodactylum tricornutum. Biochemistry 2007, 46, 9813–9822. (60) Sun, H.; Tsunenari, T.; Yau, K.-W.; Nathans, J. The vitelliform macular dystrophy protein defines a new family of chloride channels. Proc. Natl. Acad. Sci. U. S. A. 2002, 99 (6), 4008–4013. (61) Bailleul, B.; Rogato, A.; de Martino, A.; Coesel, S.; Cardol, P.; Bowler, C.; Falciatore, A.; Finazzi, G. An atypical member of the lightharvesting complex stress-related protein family modulates diatom responses to light. Proc. Natl. Acad. Sci. U. S. A. 2010, 107 (42), 18214–18219. (62) Zhu, S.-H.; Green, B. R. Photoprotection in the diatom Thalassiosira pseudonana: Role of LI818-like proteins in response to high light stress. Biochim. Biophys. Acta 2010, 1797, 1449–1457. (63) Savard, F.; Richard, C.; Guertin, M. The Chlamydomonas reinhardtii L1818 gene represents a distant relative of the cabl/ll genes that is regulated during the cell cycle and in response to illumination. Plant Mol. Biol. 1996, 32, 461–473. (64) Richard, C.; Ouellet, H.; Guertin, M. Characterization of the LI818 polypeptide from the green unicellular alga Chlamydomonas reinhardtii. Plant Mol. Biol. 2000, 42, 303–316. (65) Beer, A.; Gundermann, K.; Beckmann, J.; B€uchel, C. Subunit composition and pigmentation of fucoxanthin-chlorophyll proteins in diatoms: evidence for a subunit involved in diadinoxanthin and diatoxanthin binding. Biochemistry 2006, 45, 13046–13053. 5352

dx.doi.org/10.1021/pr200600f |J. Proteome Res. 2011, 10, 5338–5353

Journal of Proteome Research

ARTICLE

(66) Kilian, O.; Kroth, P. G. Identification and characterization of a new conserved motif within the presequence of proteins targeted into complex diatom plastids. Plant J. 2005, 41, 175–183. (67) Emanuelsson, O.; Brunak, S.; von Heijne, G.; Nielsen, H. Locating proteins in the cell using TargetP, SignalP and related tools. Nat. Prot. 2007, 2 (4), 953–971.

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