c-binding protein from the diatom, Chaetoceros gracilis

Biochimica et Biophysica Acta 1817 (2012) 2110–2117

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Proteases are associated with a minor fucoxanthin chlorophyll a/c-binding protein from the diatom, Chaetoceros gracilis Ryo Nagao a,⁎, Tatsuya Tomo b, c, Eri Noguchi b, Takehiro Suzuki d, Akinori Okumura e, Rei Narikawa a, c, Isao Enami b, Masahiko Ikeuchi a a

Department of Life Sciences (Biology), Graduate School of Art and Sciences, University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo, 153-8902, Japan Department of Biology, Faculty of Science, Tokyo University of Science, Kagurazaka 1-3, Shinjuku-ku, Tokyo, 162-8601, Japan PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan d Biomolecular Characterization Team, RIKEN Advanced Science Institute, Hirosawa 2-1, Wako, Saitama 351-0198, Japan e Department of Integrated Sciences in Physics and Biology, College of Humanities and Sciences, Nihon University, 3-25-40 Sakurajosui, Setagaya-ku, Tokyo 156-8550, Japan b c

a r t i c l e

i n f o

Article history: Received 15 June 2012 Received in revised form 21 August 2012 Accepted 27 August 2012 Available online 3 September 2012 Keywords: FCP Photosystem Protease Thylakoid

a b s t r a c t We previously showed that most subunits in the oxygen-evolving photosystem II (PSII) preparation from the diatom Chaetoceros gracilis are proteolytically unstable. Here, we focused on identifying the proteases that cleave PSII subunits in thylakoid membranes. Major PSII subunits and fucoxanthin chlorophyll (Chl) a/c‐binding proteins (FCPs) were specifically degraded in thylakoid membranes. The PSI subunits, PsaA and PsaB, were slowly degraded, and cytochrome f was barely degraded. Using zymography, proteolytic activities for three metalloproteases (116, 83, and 75 kDa) and one serine protease (156 kDa) were detected in thylakoid membranes. Two FCP fractions (FCP-A and FCP-B/C) and a photosystem fraction were separated by sucrose gradient centrifugation using dodecyl maltoside‐solubilized thylakoids. The FCP-A fraction featured enriched Chl c compared with the bulk of FCP-B/C. Zymography revealed that 116, 83, and 94 kDa metalloproteases were mostly in the FCP-A fraction along with the 156 kDa serine protease. When solubilized thylakoids were separated with clear-native PAGE, zymography detected only the 83 kDa metalloprotease in the FCP-A band. Because FCP-A is selectively associated with PSII, these FCP-A-associated metalloproteases and serine protease may be responsible for the proteolytic degradation of FCPs and PSII in thylakoid membranes. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Diatoms are unicellular photosynthetic eukaryotes responsible for approximately 20% of the global carbon fixation [1]. They are likely to have emerged following a secondary endosymbiotic process between a photosynthetic eukaryote, most likely red algal-like, and a heterotrophic eukaryote [2]. Whole genome sequences are available for two diatoms, Thalassiosira pseudonana and Phaeodactylum tricornutum [3,4]. Bioinformatic analyses have revealed the photosynthetic apparatus and their biosynthetic pathways in diatoms [5–8]. Molecular studies are now emerging, thus allowing diatoms as a new model organism for photosynthesis in addition to plants, green algae and cyanobacteria [9,10].

Abbreviations: Bis-Tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane; Chl, chlorophyll; CN-PAGE, clear-native PAGE; cyt, cytochrome; FCP, fucoxanthin chlorophyll a/c-binding protein; LDS, lithium lauryl sulfate; PS, Photosystem; SGC, sucrose gradient centrifugation ⁎ Corresponding author at: Department of Integrated Sciences in Physics and Biology, College of Humanities and Sciences, Nihon University, 3-25-40 Sakurajosui, Setagaya-ku, Tokyo 156-8550, Japan. Fax: +81 3 5317 9432. E-mail address: [email protected] (R. Nagao). 0005-2728/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbabio.2012.08.005

The oxygen-evolving PSII is one of the key complex in the photosynthetic apparatus and is a target for dynamic regulation under variable environmental conditions in nature. Biochemical analyses revealed that a novel extrinsic protein of ~13.5 kDa, which was later termed Psb31, was associated with the oxygen-evolving PSII from a diatom, Chaetoceros gracilis [11–13]. Release-reconstitution experiments demonstrated that Psb31 is essential for optimal oxygen evolution [14]. Genome analysis confirmed that Psb31 and four extrinsic proteins are shared with other diatoms and red algae, supporting the red algal lineage [12]. Of these, the diatom C. gracilis is particularly suitable for isolation and characterization of the oxygen-evolving PSII complex. However, it turned out that the crude preparation of the oxygenevolving PSII complex (crude PSII) was remarkably unstable; rapid inactivation of the oxygen-evolving activity, pigment bleaching, and protein degradation were observed [13]. Proteolytic degradation of thylakoid proteins has been extensively studied in plants in vivo and in vitro [15–17]. Of these, D1 protein of PSII is the major target of rapid turnover during photodamage and subsequent recovery processes [18,19]. Specific proteases (FtsH and Deg) involved in the rapid turnover of D1 have been studied using Arabidopsis and some cyanobacteria [20,21]. Many other proteases (e.g., Clp, Lon, CtpA) have also been suggested to be involved in

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Fig. 1. Effects of inhibitors on proteolytic degradation in thylakoid membranes. Thylakoid membranes (0.5 mg Chl ml−1) were incubated at 25 °C for 0, 3, 6, 9, or 24 h in the dark in the absence or presence of 5 mM EDTA, 1 mM PMSF, or 5 mM EDTA/1 mM PMSF. Treated thylakoids (5 μg Chl) were subjected to SDS-PAGE.

regulating protein turnover in chloroplasts [20]. However, no biochemical analysis of proteases in terms of degradation of chloroplast proteins in diatoms has been reported. In this study, we focused on identifying the proteases that are associated with thylakoid membranes of C. gracilis. We identified them by zymography and detergent fractionation. Together with specific degradation of PSII and FCP polypeptides, we discuss the role of these proteases.

Tris-HCl (pH 8.0) for 24 h at 25 °C. The casein in the gel was stained with CBB to detect negative bands at renatured proteases on a background of uniform staining. To express the bands more clearly, a scanned image of the casein gel was inverted. The standard molecular marker proteins were separated next to the sample lanes in the casein gel to estimate the size of negative bands after appropriate destaining.

2.5. Immunological assays 2. Materials and methods 2.1. Growth and isolation of thylakoid membranes The marine centric diatom, C. gracilis Schütt (UTEX LB 2658), was grown in artificial seawater [11]. Thylakoid membranes were prepared according to Nagao et al. [11,13] and suspended in a medium containing 1 M betaine and 50 mM MES-NaOH (pH 6.5). Chl a and c concentrations were determined in 90% acetone using the equation of Jeffrey and Humphrey [22]. 2.2. Proteolytic degradation Proteolytic degradation was performed at 25 °C in the dark in the absence or presence of 5 mM EDTA and/or 1 mM PMSF. Reactions were stopped by addition of lithium lauryl sulfate (LDS) and dithiothreitol as mentioned below. Some details of the condition are provided in each figure legend. 2.3. SDS-PAGE Samples were solubilized with 5% LDS and 75 mM dithiothreitol for 30 min on ice. The solubilized samples were subjected to SDS-PAGE using a gradient of 16–22% acrylamide and 7.5 M urea [23]. After electrophoresis, gels were stained with Coomassie Brilliant Blue R-250 and photographed. A standard molecular marker (Molecular Weight Marker, High Range; Wako Pure Chemical Industries, Japan) was used. 2.4. Zymography Zymography was performed as described [24,25] with some modifications. Samples were solubilized with 5% LDS for 30 min on ice and then subjected to SDS-PAGE at 4 °C through 8% polyacrylamide gels containing 1 mg ml−1 casein. Then, SDS and LDS were removed by washing the gel in 2.5% (w/v) Triton X-100 for 1 h at 4 °C, and casein was digested during subsequent incubation of the gel in 50 mM

For western blotting, proteins on the SDS-PAGE gel were transferred onto a polyvinylidene difluoride membrane and probed with specific antibodies. Horseradish peroxidase–conjugated anti-IgG was used as a secondary antibody. Chemiluminescence was detected using ECL Western Blotting Detection Reagents (GE Healthcare Biosciences, UK) and an imaging system with a LumiCube (Liponics, Japan). Specific antibodies against PsaA/B, D2, and D1 were used as described [11,26]. The antibody against cytochrome f (cyt f) from the diatom, P. tricornutum, was prepared in this study. The coding sequence of petA was amplified by PCR from genomic DNA of P. tricornutum, with primers 5′-CGCATATGTATCCAGTTTTTGCACAACA-3′ and 5′-GGGAATT CTTAGAAGTTTAATTCTGCAGC-3′; the PCR product was cloned into the pET28a vector (Novagen, USA) at NdeI and EcoRI sites (underlined). The cyt f protein was expressed in Escherichia coli BL21 (DE3) after induction with 1 mM isopropylthio-β-galactoside and recovered in inclusion bodies. Proteins in inclusion bodies were solubilized with 6 M urea for 20 min on ice. After centrifugation at 40,000 ×g for 10 min, the supernatant was loaded onto a Ni affinity column (ProBond™ Resin; Invitrogen, USA) for purification. The antibody against cyt f was prepared in rabbit (Iwaki, Japan).

2.6. Sucrose gradient centrifugation (SGC) Thylakoid membranes were solubilized with 4% dodecyl maltoside (DM) at 1.0 mg Chl ml−1 for 10 min on ice in the dark and clarified by centrifugation at 40,000 ×g for 10 min. The supernatant was diluted ten-fold with 50 mM MES-NaOH (pH 6.5), and 100 μg Chl was loaded onto a linear sucrose gradient of 10–20% (w/v) in a medium containing 5 mM CaCl2, 50 mM MES-NaOH (pH 6.0), and 0.04% DM (buffer A) in a total volume of 30 ml. After centrifugation at 103,200 ×g for 18 h (P28S rotor; Hitachi, Japan), fractions were obtained. Each fraction was washed twice with an equal volume of buffer A containing 15% sucrose and concentrated using a 50 kDa cut-off filter (Amicon Ultra; Millipore, USA) at 2000 ×g.

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dilutions at 0 h

2.7. Absorption spectra

100%

Absorption spectra were measured at 25 °C using a V-660 spectrophotometer (JASCO, Japan) and normalized to the absorption at the maximum of the Qy peak.

50%

dark incubation

25%

0

3

6

9

24 h

D1 D2

2.8. Clear-native PAGE (CN-PAGE) PsaA/B

SF

n

/P TA ED

PM

TA ED

SF

M

tio di

We examined proteolytic degradation in thylakoid membranes during incubation at 25 °C in the dark in the presence or absence of protease inhibitors (Fig. 1). Thylakoid proteins were identified based on previous reports [11,28], and FCPs were termed FCP-A, FCP-B, and FCP-C in this study (see Supplemental Fig. S1). In the absence of protease inhibitors, FCP-A and PSII subunits (CP47, CP43, PsbO, D2, and D1) mostly disappeared during dark incubation, whereas FCP-B and -C mostly remained. The degradation of FCP-A, -B, -C, and PSII subunits was mostly suppressed in the presence of the metalloprotease inhibitor, EDTA, while the degradation of PSII subunits was only partly suppressed in the presence of the serine protease inhibitor, PMSF. Both EDTA and PMSF in combination suppressed even more degradation, although some degradation was still observed. The degradation of PSII, PSI, and cyt b6 f subunits in thylakoid membranes was examined by western blotting (Fig. 2). Rapid degradation of D1 and D2 was observed during dark incubation within 9 h, and these proteins disappeared by 24 h. PsaA/B subunits were also degraded, although more slowly than D1 and D2 subunits. Cyt f was barely degraded even after 24 h. These results together with the above SDS-PAGE results indicate that PSII and FCP subunits were preferentially degraded in thylakoid membranes.

FCP-B/C. The green-colored fraction III was composed mainly of PSI and PSII subunits and a small amount of FCP-A. A room-temperature absorption spectrum of fraction I (Fig. 5) showed major peak of Chl a at approximately 671.8 nm, a small peak of Chl c at approximately 635.4 nm, and broad shoulders at 450–500 nm (Chl c and diadinoxanthin) and 500–570 nm (fucoxanthin), consistent with known spectra [13,29–31]. These peaks are quite similar to those of major FCPs from other diatom [29]. The spectrum of fraction II (dotted line) resembled that of fraction I (solid line), especially a major peak of Chl a at approximately 670.8 nm. However, the Chl c peak at 637.0 nm was more prominent and the shoulder at 450–490 nm was higher than that of fraction I; the shoulder at 500–570 nm was slightly lower than that of fraction I. These

ad

3.1. Proteolytic degradation of thylakoid proteins

Fig. 2. Degradation time course of subunits of PSII, PSI, and cyt b6f complexes. The incubated thylakoids (1 μg Chl) were subjected to SDS-PAGE, and subunit proteins were detected by western blotting using antibodies raised against D1, D2, PsaA/B, and cyt f. A dilution series (100%, 50%, and 25%) of thylakoids at 0 h, corresponding to 1.0, 0.5, and 0.25 μg Chl, was used as a calibration standard.

o

3. Results

cyt f

N

CN-PAGE was performed as described [27] with some modifications. Thylakoid membranes were suspended in a medium containing 50 mM bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris-HCl) (pH 7.0), 500 mM 6-aminocaproic acid, and 10% glycerol, followed by solubilization in 4% DM at 1.0 mg Chl ml−1 for 10 min on ice in the dark. After centrifugation at 40,000 ×g for 10 min, the supernatant was applied to a 4–18% polyacrylamide gel. Each sample containing 5 μg Chl was loaded per lane. A standard molecular marker (NativeMark™; Invitrogen) was used. Electrophoresis was carried out at 4 °C in electrophoresis buffer containing 50 mM Tricine and 50 mM Bis-Tris-HCl (pH 7.0), in which 0.02% sodium deoxycholate and 0.02% (w/v) Triton X-100 were added in the cathode buffer. The lanes were cut out and subjected to SDS-PAGE.

kDa 200

A B C D

116

79

42 3.2. Detection of proteolytic activities with zymography Proteases in thylakoid membranes were detected with zymography using casein in SDS-PAGE (Fig. 3). Four bands with activity (marked A–D) were visualized, and the apparent molecular masses of proteases A–D were estimated as approximately 156, 116, 83, and 75 kDa, respectively. The active bands B–D disappeared in the presence of EDTA, whereas band A disappeared in the presence of PMSF. All bands disappeared in the presence of both EDTA and PMSF. These results suggest that proteases B–D are metalloproteases and protease A is a serine protease.

30

3.3. Proteases in pigment–protein complexes separated by sucrose gradient centrifugation Pigment–protein complexes were separated by SGC after solubilization of thylakoid membranes with DM (Fig. 4). The brown-colored fraction I was composed exclusively of FCP-B/C. The pale brown-colored fraction II was composed mainly of FCP-A and a small amount of

Fig. 3. Proteolytic activity in thylakoid membranes: zymography of SDS-PAGE. Thylakoids (5 μg Chl) were subjected to SDS-PAGE containing 1 mg ml−1 casein. After removal of SDS, in-gel digestion of casein was done for 24 h at 25 °C in the absence or presence of 5 mM EDTA, 1 mM PMSF, or 5 mM EDTA/1 mM PMSF. Letters indicate the identified proteases. See the text for details.

n

II ct io fra

n

I ct io

n fra

ct io fra

kDa

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III

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116 79

PsaA/B CP47 CP43

fractionI 42

fraction II

D2 D1

30

fraction III

17

FCP-B FCP-C

FCP-A

Fig. 4. Separation of pigment–protein complexes by SGC and their polypeptide compositions. Fractions I (1.0 μg Chl), II (1.0 μg Chl), and III (2.0 μg Chl) were subjected to SDS-PAGE.

results suggest that Chl c (and possibly diadinoxanthin, too) are more enriched in FCP-A than FCP-B/C. On the other hand, fraction III (dashed line) showed an absorption maximum at 676.2 nm owing to Chl a originated in the mixture of PSII, FCP-A, and PSI, and a shoulder at 450–490 nm owing to Chl c originated in FCP-A. We examined the proteolytic degradation of the SGC fractions during incubation at 25 °C in the dark in the presence or absence of protease inhibitors (Fig. 6). In fraction I, FCP-B/C were partially degraded in the absence of protease inhibitors. Degradation was slightly suppressed in the presence of EDTA, whereas it was not visibly suppressed in the presence of PMSF. In fraction II, FCP-A was mostly degraded in the absence of protease inhibitors. Degradation was partially suppressed in the presence of EDTA or PMSF and was mostly suppressed in the presence of both EDTA and PMSF. In fraction III, FCP-A and PSII subunits were degraded, but PsaA/B were only slightly degraded in the absence of protease inhibitors. Degradation of FCP-A

and PSII subunits was slightly suppressed in the presence of EDTA, while it was only slightly suppressed in the presence of PMSF. Prominent protease activities at four bands were observed in fraction II, although some were detected in fractions I and II (Fig. 7). The four active bands corresponded to approximately 156, 116, 94, and 83 kDa. Three bands disappeared in the presence of EDTA, and the other band disappeared in the presence of PMSF. These results indicate that the band at 156 kDa is a serine protease, and the bands at 116 and 83 kDa are metalloproteases B and C. The band at 94 kDa does not correspond to metalloprotease D (75 kDa) and was named metalloprotease E. In fraction III, these weak protease bands were visualized at approximately 156, 116, and 83 kDa. Based on the sensitivity to inhibitors, these proteases were identified as proteases A, B, and C. Because fraction III retained some FCP-A, these proteases may be still associated with FCP-A and involved in the degradation of FCP-A and PSII. In fraction I, two bands were detected and assigned as metalloproteases C and E, based on the sensitivity to EDTA. The metalloprotease D of 75 kDa, which was detected in thylakoid membranes, was not detected in any fraction.

Absorbance (a.u.)

3.4. Proteases in pigment–protein complexes separated with CN-PAGE

350

400

450

500

550

600

650

700

750

Wavelength (nm) Fig. 5. Room-temperature absorption spectra of fractions I (solid line), II (dotted line), and III (dashed line). The spectra were normalized to the absorption at the maximum of the Qy peak.

Pigment–protein complexes were separated with CN-PAGE after solubilization of thylakoid membranes with DM. Four pigmentcontaining bands (marked I–IV) were obtained (Fig. 8A), and their polypeptides were resolved by two-dimensional CN-/SDS-PAGE (Fig. 8B). Band I was composed mainly of PSI subunits. Band II was composed of PSII subunits. The pale brown‐colored band III was composed of FCP-A, and the brown-colored band IV was composed of FCP-B/C. Proteolytic activities were examined with zymography of twodimensional CN-/SDS-PAGE (Fig. 9). Only one spot with activity was visualized from FCP-A (band III), and its apparent molecular mass was estimated as approximately 83 kDa (see Supplemental Fig. S2). This spot disappeared in the presence of EDTA but was unchanged in the presence of PMSF. These results suggest that metalloprotease C is tightly associated with FCP-A. On the other hand, no proteolytic activity was

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EDTA/PMSF

PMSF

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PsaA/B CP47 CP43 D2 D1 FCP-A

42 30

17

No addition

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EDTA/PMSF

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fraction III 24 h

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No addition

EDTA/PMSF

EDTA

fraction II 24 h

0h

PMSF

EDTA

kDa

fraction I 24 h No addition

No addition

0h

No addition

2114

Fig. 6. Effects of inhibitors on proteolytic degradation in fractions I–III. Each fraction (0.1 mg Chl ml−1) was incubated for 24 h in the dark in the absence or presence of 5 mM EDTA, 1 mM PMSF, or 5 mM EDTA/1 mM PMSF. Treated fractions I (1.0 μg Chl), II (1.0 μg Chl), and III (2.0 μg Chl) were subjected to SDS-PAGE.

and they were partially recovered in the photosystem-enriched fraction. Two-dimensional CN-/SDS-PAGE of the DM-solubilized thylakoids suggested that at least protease C is specifically associated with FCP-A oligomers. Further fractionation and/or sensitive detection would allow identification of protease C, which is now in progress. FCPs are major light-harvesting antennae encoded by a multigene family in the nuclear genome [32–34]. Nonetheless, functional differentiation of FCPs has attracted little attention except for PSI-specific FCPs in C. gracilis [28] and Cyclotella meneghiniana [35]. Previously, we reported crude and purified oxygen-evolving PSII particles [11,13]. Although FCPs were not isolated in those studies, SDS-PAGE profiles allowed us to determine that FCP-A is a component of crude PSII. FCP-A is also clearly absent from the PSI/FCP supercomplex [28] (see also Supplemental Fig. S1). These results suggest that a supercomplex of PSII and FCP-A was recovered in the photosystem-enriched fraction of SGC. The

116 79

E C

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B E C

B

SF M /P TA ED

o N

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SF

ad

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/P TA ED

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TA

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n o N

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ad di tio

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M SF

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ED TA

kDa 200

PM

N o

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di tio ED n TA

fraction I

PM

To gain insight into protein stabilization and turnover in thylakoids, we detected proteases in thylakoid membranes of C. gracilis using zymography and detergent fractionation. At least four proteases were detected in thylakoid membranes: serine protease A (156 kDa), metalloprotease B (116 kDa), metalloprotease C (83 kDa), and metalloprotease D (75 kDa). When thylakoid membranes were solubilized with DM and fractionated with SGC, the major 83 kDa metalloprotease was in the FCP-A‐enriched fraction along with the 156 kDa serine protease and the 116 and 94 kDa metalloproteases,

TA

4. Discussion

ED

detected from band I, II, or IV. Proteases A, B, D, and E observed in thylakoid membranes or SGC fractions were not detected with CN-PAGE. These proteases may have diffused near the front in the firstdimension CN-PAGE or may have been inactivated during CN-PAGE.

C

42

30

Fig. 7. Proteolytic activity of the SGC fractions I–III: zymography of SDS-PAGE. After removal of SDS, in-gel digestion of casein was done for 24 h at 25 °C in the absence or presence of 5 mM EDTA, 1 mM PMSF, or 5 mM EDTA/1 mM PMSF. Letters indicate the identified proteases. See the text for details.

2115

IV (FCP-B/C)

kDa

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B I (PSI)

A

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1236 1048

I (PSI) 720

II (PSII) 480

242

III (FCP-A)

146

IV (FCP-B/C) 66

20

Fig. 8. Separation of pigment–protein complexes with CN-PAGE (A) and two-dimensional CN-/SDS-PAGE (B). Representative spots are PsaA/B of PSI (square), CP47, CP43, D2, and D1 of PSII (triangles), FCP-A (diamond), and FCP-B/C (circles).

FtsHs. Degs are ATP-independent serine proteases with molecular masses ranging from approximately 30 to 100 kDa [42–48]. This size range is much smaller than that of serine protease A, which is 156 kDa. These results strongly support the idea that the proteases described here may not correspond to FtsHs or Degs. Here we report for the first time a purification of FCP-A, which is a minor FCP in C. gracilis. FCP-A was exclusively isolated as an oligomeric complex in SGC and CN-PAGE after solubilization with DM. The absorption spectrum of FCP-A showed the more prominent peak of Chl c in addition to the expected peaks of Chl a and fucoxanthin. These features are in contrast to the bulk FCP-B/C. The

IV (FCP-B/C)

II (PSII)

I (PSI)

PMSF IV (FCP-B/C)

III (FCP-A)

II (PSII)

I (PSI)

EDTA IV (FCP-B/C)

III (FCP-A)

II (PSII)

I (PSI)

No addition

III (FCP-A)

degradation of FCP-A and PSII polypeptides in thylakoid membranes and crude PSII is very likely due to the proteases that are mainly associated with FCP-A. Assuming such a localization, PSI polypeptides were likely not rigorously degraded. The proteases FtsH and Deg are responsible for specific proteolysis of damaged D1 during exposure to strong light. FtsHs are ATPdependent metalloproteases with molecular masses of approximately 60–80 kDa [36–41]. We examined the effects of ATP on the proteolysis of thylakoid membranes. The results showed almost no enhanced proteolysis in the presence of ATP (see Supplemental Fig. S3), suggesting that metalloproteases B, C, D, and E are not related to

Fig. 9. Proteolytic activity of thylakoid membranes: zymography of two-dimensional CN-/SDS-PAGE. After removal of SDS, in-gel digestion of casein was done for 24 h at 25 °C in the absence or presence of 5 mM EDTA or 1 mM PMSF.

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organization and absorption spectrum of FCP-B/C are very similar to those of major FCP of other diatoms such as C. meneghiniana [29,31,49], Cyclotella cryptica [50], and P. tricornutum [30,51,52]. However, it remains unsolved whether the minor antenna such as FCP-A is conserved in other diatom or not. Previously, we reported that FCP-A was loosely associated with oxygen-evolving PSII particles [13]. In addition, the FCP-A fraction retained at least four proteases. These results suggest that FCP-A may play multiple roles in light harvesting and/or protein turnover of PSII in the thylakoid of C. gracilis and possibly other diatoms. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.bbabio.2012.08.005. Acknowledgments This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education of Japan (No. 21570038, 22370017, 24370025 to T.T., No. 20608001 to Rei.N., No. 17657013, 20370018, 21657013 to M.I.), by a Research Fellowship to Ryo.N. from the Japan Society for the Promotion of Science, by a grant from JST PRESTO to T.T. and Rei.N., by a grant from Australian Research Council's Discovery Projects funding scheme (project number DP12101360) to T.T., by a grant from JST CREST to M.I., and by the Global COE program (From the Earth to ‘Earths’) to M.I. References [1] C.B. Field, M.J. Behrenfeld, J.T. Randerson, P. Falkowski, Primary production of the biosphere: integrating terrestrial and oceanic components, Science 281 (1998) 237–240. [2] P.G. Falkowski, M.E. Katz, A.H. Knoll, A. Quigg, J.A. Raven, O. Schofield, F.J.R. Taylor, The evolution of modern eukaryotic phytoplankton, Science 305 (2004) 354–360. [3] E.V. Armbrust, J.A. Berges, C. Bowler, B.R. 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