Proteomics Analysis of Oil Body-Associated Proteins in the

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Proteomics analysis of oil body-associated proteins in the oleaginous diatom Daisuke Nojima, Tomoko Yoshino, Yoshiaki Maeda, Masayoshi Tanaka, Michiko Nemoto, and Tsuyoshi Tanaka J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr4004085 • Publication Date (Web): 23 Jul 2013 Downloaded from http://pubs.acs.org on July 29, 2013

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Journal of Proteome Research

Proteomics analysis of oil body-associated proteins in the oleaginous diatom

Daisuke Nojima a, b, Tomoko Yoshinoa, Yoshiaki Maedaa, Masayoshi Tanakaa,b, Michiko Nemotoa, Tsuyoshi Tanakaa,b*

a

Institute of Engineering, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, Tokyo, 184-8588, Japan b

JST, CREST, 5, Sanbancho, Chiyoda-ku, Tokyo, 102-0075, Japan

*Author for correspondence Tel: +81-42-388-7021 Fax: +81-42-385-7713 E-mail: [email protected]

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Abstract For biodiesel production from microalgae, it is desirable to understand the entire triacylglycerol (TAG) metabolism. TAG accumulation occurs in oil bodies, and although oil body-associated proteins could play important roles in TAG metabolism, only a few microalgal species have been studied by a comprehensive analysis. Diatoms are microalgae that are promising producers of biodiesel, on which such proteomics analysis has not been conducted to date. Herein, we identified oil body-associated proteins in the oleaginous diatom Fistulifera sp. strain JPCC DA0580. The oil body fraction was separated by cell disruption with beads beating and subsequent ultracentrifugation. Contaminating factors could be removed by comparing proteins from the oil body and the soluble fractions. This novel strategy successfully revealed fifteen proteins as oil body-associated protein candidates. Among them, two proteins, which were parts of proteins predicted to have transmembrane domains, were indeed confirmed to specifically localize to the oil bodies in this strain by observation of GFP fusion proteins. One (predicted to be a potassium channel) was also detected from the ER, suggesting that oil bodies might originate from the ER. By utilizing this novel subtraction method, we succeeded in identifying the oil body-associated proteins in the diatom for the first time.

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Keywords: Marine diatom Fistulifera sp. strain JPCC DA0580 Oil body-associated proteins Oil body proteomics Origin of oil body Endoplasmic reticulum Chloroplast Biodiesel production

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Introduction With an increased demand for a sustainable energy supply, biofuel production has attracted much attention. Microalgal biodiesel production is an emerging technology in such demand because of the organisms’ great features for this purpose (e.g., global carbon dioxide fixation, no competition for food, much higher biomass yield than higher plants, and oil accumulation at a high level inside the cells1-5). Most recent studies have demonstrated that candidate species for biofuel production are diverse and could be found in several microalgal divisions including Chlorophyta, Heterokontophyta (including Bacillariophyceae, the so-called diatom, and Eustigmatophyceae), Haptophyta, Rhodophyta and Dinoflagellata2, 6-8. Among them, several oleaginous species can accumulate more than 60% of triacylglycerol (TAG) in dry cell weight, and such promising oil producers have been intensively studied to understand the TAG metabolism towards the practical applications of microalgal biofuel production9. Although current studies have focused more on the synthesis pathways for fatty acids and TAG (Kennedy pathway)10, 11, storage and subsequent degradation of TAG in microalgae should be studied for elucidation of the whole context of TAG utilization, and such studies have just launched by analyzing the proteins closely attached around the oil droplets. The preceding studies in higher plants like Arabidopsis thaliana demonstrated that TAG accumulation resulted in formation of particular organelles, the so-called oil bodies (also known as oleosomes, lipid particles or lipid droplets), which were surrounded by a phospholipid monolayer12. Proteomics analysis of the oil bodies revealed that some proteins are specifically associated with the 4


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oil bodies and are considered to play important roles in accumulating and degrading TAGs. Such proteins have been isolated from not only higher plants but also animals, fungi, and microalgae 13. To date, the oil body-associated proteins were found in 4 Chlorophyta microalgae: Chlamydomonas reinhardtii, Haematococcus pluvialis, Dunaliella sp., Chlorella sp. and in one Eustigmatophyceae: Nannochloropsis sp.14-20. Despite the wide diversity of oleaginous microalgae, oil body-associated proteins from other taxonomic groups including diatoms, one of the most promising groups for biodiesel production, remains unknown.

In animals and higher plants, some proteins were identified as major proteins in the oil bodies of individual species. Perilipins, oleosins21, steroleosins22 and caleosins23 were such examples and are considered to play a critical role in determining the size and stability of oil bodies in each species. In microalgae, there are different types of major proteins termed major lipid droplet protein (MLDP) for C. reinhardtii18,17 and Dunaliella sp.20 , lipid droplet surface protein (LDSP) for Nannochloropsis sp.15 and oil globule protein for H. pluvialis16. Most of these major microalgal proteins in oil bodies do not show high sequence-similarity with those in higher plants excepting a rare example of caleosin-like protein found in Chlorella sp.23.

Other than major proteins in the oil bodies, less abundant proteins could also play roles in lipid metabolism. For instance, phospholipase D1 (PLD1) and extracellular signal-regulated kinase 2 (ERK2) are known to regulate cytosolic oil body formation in animal cells. Moreover, in the higher plant A.thaliana, a patatin-like TAG lipase was detected on the oil bodies24. Numerous such 5



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examples have been reported, implying that characterization of major proteins in the oil bodies is not enough to fully understand the metabolism of TAG utilization.

Another important issue exists in the oil body research, which is that it remains unclear whether oil body membranes in microalgae are derived from the endoplasmic reticulum (ER) or chloroplasts. Oil bodies are classified according to the location in which the organelle arises; i.e., in the cytoplasm or in chloroplasts. In general, cytoplasmic oil bodies are recognized to originate from the ER membrane25. On the other hand, the oil bodies are localized in both the cytoplasm and chloroplasts in green microalgae, such as C. reinhardtii10, 11 and D. bardawil26. In such cases, the oil body membrane in chloroplasts could be derived from thylakoid membranes. However, these hypotheses are still debated. In such circumstances, a comprehensive analysis of oil body-associated proteins could provide clues to address this issue because the protein profiles of oil bodies would become evidences to estimate the origin of oil body membranes.

Despite the importance of the oil body-associated proteins, such studies for microalgae remained limited as mentioned above. This could be due to the complicated cellular structure resulting from symbiosis that makes it extremely difficult to purify oil bodies from the ER and chloroplast fractions. Therefore, although the proteomics approach itself is recognized as a powerful tool for whole cell analysis during oil production27, 28, fractionation of specific compartments without any contamination has not been achieved even for green algae, which have relatively simple cellular structures16-19. Diatoms have never been subjected to proteomics analysis for oil bodies because they have more 6


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complicated membrane systems than green algae, owing to the evolutional history with secondary symbiosis. Gentle disruption strategies, which were sometimes effective with green algae18, 20, are no match for the stiff frustules composed of SiO2; these features make it almost impossible to purely isolate oil bodies from diatoms.

To overcome these difficulties and acquire insights on the TAG metabolism and origin of oil bodies in diatoms, we proposed to employ a comparative strategy in order to distinguish the oil body-specific proteins and other contaminations. As a proof-of-concept, the oil body fraction and the soluble fraction from an oleaginous diatom, Fistulifera sp. strain JPCC DA05803 were separated by ultracentrifugation, followed by protein identification using nanoLC-MS/MS. By comparing proteins identified from these fractions, 22 proteins were selected. After removing the contaminants from other compartments like the chloroplasts, mitochondria and nucleus to identify 15 oil body-specific proteins, further narrowing down was taken place based on in silico prediction of transmembrane domains. Finally, we obtained 5 candidate proteins which could specifically and strongly associate with oil bodies in this strain. The GFP-fusion technique was utilized to confirm the localization of the candidate proteins in vivo, and from these observations, we discuss the TAG metabolism and origin of oil body membranes. To the best of our knowledge, this is the first report with respect to oil body-associated proteins in a diatom. Furthermore, new discoveries in this study could open the door for elucidation of TAG metabolism in oleaginous diatoms.

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Materials and Methods

Strain and culture conditions

The stock culture of the marine oleaginous diatom, Fistulifera sp. strain JPCC DA0580 was maintained in Erlenmeyer flasks in 10f medium (1.5 g NaNO3, 120 mg Na2HPO4 · 2H2O, 10 µg vitamin B12, 10 µg biotin, 2 mg Thiamine HCl, 200 mg Na2SiO3 · 9H2O, 88 mg Na2-EDTA, 63.2 mg FeCl3 · 6H2O, 240 µg CoSO4 · 5H2O, 420 µg ZnSO4 · 7H2O, 3.6 mg MnCl2 · 4H2O, 1.4 mg CuSO4 · 5H2O, and 140 µg Na2MoO4 · 2H2O) dissolved in a liter of artificial seawater (NaCl 22.1 g/L, MgCl2·6H2O 9.9 g/L, CaCl2·2H2O 1.5 g/L, Na2SO4 3.9 g/L, KCl 0.61 g/L, NaHCO3 0.19 g/L, KBr 96 mg/L, Na2B4O7·10H2O 78 mg/L, SrCl2 13 mg/L, NaF 3 mg/L, LiCl 1mg/L, KI 81 µg/L, MnCl2·4H2O 0.6 µg/L, CoCl2·6H2O 2 µg/L, AlCl3·6H2O 8 µg/L, FeCl3·6H2O 5 µg/L, Na2WO4·2H2O 2 µg/L, (NH4)6Mo7O24·4H2O 18 µg/L), which contains 20-fold more nutrition components than f/2 medium, a culture medium commonly used for diatoms29. For every experiment, the pre-culture was prepared by diluting stock culture into the freshly prepared 10f medium (200 mL, 1.0×106 cells/mL) and incubated for 1 week. After the pre-culturing, two-phase cultivation was performed in order to induce neutral lipid accumulation in microalgal cells. As the first incubation phase, pre-culture was transferred into a flat-shape flask including 10f medium (1.5 L) with an initial cell concentration of 1.0×106 cells/mL and cultured for about 72 h. Subsequently microalgal cells were collected by centrifugation at 8,000 ×g for 10 min. As the second incubation phase, the collected cells were 8


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resuspended in artificial seawater without any nutrient supplementation and incubated for 96 h. The temperature was maintained at 25 ± 1 °C using temperature-controlled baths. Continuous illumination was applied at 200 µmol/m2·s to the flat-shaped flasks using cool fluorescent tubes from one direction. Illumination intensity was measured using illuminometers. The cultures were bubbled with sterile air containing 2% CO2. BODIPY staining BODIPY stock solution (25 µg/mL in dimethyl sulfoxide) was diluted 50-fold with deionized water. The diluted solution was mixed into algal culture at 1 to 99 volume ratio, and the mixture was incubated at room temperature, light shielded for 10 min. Separation of oil body proteins from Fistulifera sp. strain JPCC DA0580 Oil bodies were separated based on previously described methods18 with some modifications. Briefly, microalgal cells were harvested by centrifugation at 8,500 ×g for 10 min, and resuspended in Breaking buffer (50 mM HEPES, pH7.5; 5 mM MgCl2, 5 mM KCl, 0.5 M sucrose, complete protease inhibitor tablets (Roche Diagnostics Japan, Tokyo, Japan)). The cells were disrupted by Multi-Beads Shocker (Yasui-Kikai, Osaka, Japan) at 4˚C for 15 min using 0.5 mm zirconia silica beads. Another method to break the cells was sonication at 30 W for 30 min in an ice-cold bath. The homogenates prepared by either method were then centrifuged at 1,000 ×g for 5 min to remove solid materials (e.g., frustules and silica beads). The supernatants were further centrifuged at 100,000 ×g for 60 min. From the samples prepared by bead beating, the oil body fraction was collected by using 9



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tweezers and resuspended in deionized water. Four volumes of cold acetone were added to the oil body suspension to induce the phase separation. The proteins that were accumulated at the oil-water interface were carefully collected by using a pipette and resuspended in 1% sodium dodecyl sulfate (SDS) solution. Protein concentration was determined by bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Rockford, USA) Determination of oil contents In order to extract total oil, the lyophilized cells (50 mg) were disrupted by using a mortar and a pestle. The disrupted cells were suspended in n-hexane, and the suspension was transferred to a centrifugation tube. After centrifugation (1,000 ×g, 5min, 4 ˚C), the supernatant obtained was transferred to a glass vial. n-hexane was evaporated using argon gas, and the oil was weighed. The cellular oil content was expressed as a percentage of dry cell weight (dcw) (w/w). In order to determine the oil contents included in the oil body fraction and soluble fraction separated by ultracentrifugation, both fractions were prepared as described above. The oil in the soluble fraction was extracted by adding n-hexane (1:1 volume ratio). After centrifugation (1,000 ×g, 5min, 4 ˚C), the hexane phase was recovered and the solvent was evaporated using argon gas. The oil in the oil body fraction was suspended in deionized water, and then mixed with four times its volume of acetone. After centrifugation, acetone phase was recovered and evaporated as mention above. Subsequently, the remained oil was weighted. SDS-PAGE and in-gel trypsin digestion 10


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The aqueous solution of the proteins extracted from the protein pad were mixed with SDS sample buffer (50 mM Tris-HCl, pH6.8; 1% SDS, 10% Glycerol, 0.1 M Dithiothreitol, 0.2% Bromophenol blue) and boiled at 100 ˚C for 10 min. The proteins were separated on a 12.5% acrylamide gel and stained by Bio-Safe CBB G-250 stain (Bio-rad laboratories, California, USA). The lanes containing oil body-associated proteins were excised and processed for in-gel trypsin digestion as previously described.30 MS/MS analysis by nanoLC-MS nanoLC-MS analyses were performed on an ESI-IT mass spectrometer (LCQ-DECA XP; Thermo Fisher Scientific, California, USA) coupled with a direct nanoflow LC system (DiNa; KYA Technologies, Tokyo, Japan). The extracted peptide solution was desalted in a reverse phase trap column (C18 trap column; 1 mm in length; id 0.5 mm) and resolved on a reverse phase separation column (C18 separation column; 50 mm in length; id 0.1 mm). Peptides were separated by a 70 min solvent gradient with solvent A (2% acetonitrile, 0.1% formic acid) and solvent B (80% acetonitrile, 0.1% formic acid). To obtain efficient separation of the peptides, the gradient started from 0% B for 2.1 min, followed by an increase to 8% B in 3 min, with an increase to 45% B in 30 min and a further increase to 100% B in 5 min, and remained at 100% B for the next 10 min, and returned to 0% B for 20 min. The total run time was set to 70.1 min. The flow rate was 300 nL·min-1. The full-scan mass spectra were measured from m/z 500 to 2000. MS/MS analyses were operated in the data-dependent mode where the strongest peaks detected in the MS scan were selected every 30 11



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msec, and were further analyzed with MS/MS scan. The details of MS analysis were shown as follows; the relative collision energy = 35%, dynamic exclusion parameters; repeat count = 2, repeat duration = 15, exclusion list size = 25, exclusion duration = 3, Exclusion mass width =1.5. Protein identification Peak lists were generated with the BioWorksTM 3.3 software (SEQUEST, Thermo Fisher Scientific, California, USA) from the nanoLC-MS/MS raw data as follows: peptide molecular weight range was 600-3,500; threshold was 100,000; precursor mass tolerance was 1; group scan was 1; minimum group count was 1; minimum ion count was 10; fragment ion tolerance was 1; peptides without charge were not determined; acceptable missed internal cleavage sites were equal to or less than 2. All peptide sequences were blasted against a homemade JPCC DA0580 protein database (including 20470 sequences based on whole genome information) using the SEQUEST ver. 2.0 search engine. The criterions for protein identification were cross correlation (XC) values (XC > 1.9 when peptide with a +1 charge state (z), XC > 2.2 when z = + 2, and XC > 3.75 when z = + 3, respectively). The minimal number of unique peptides to conserve a protein was over the 1 peptide. Transformation of JPCC DA0580 Expression vectors for GFP-fused putative oil body-associated proteins were constructed by using general gene fusion techniques where GFP was fused at the C-terminus of each target protein. Briefly, we utilized the neomycin phosphotransferase II expression plasmid vector with an H4 promoter (pSP-NPT/H4) constructed in our previous study31. The GAPDH promoter sequence (500 bp of 12


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GAPDH gene upstream) of strain JPCC DA0580 and fucoxanthin chlorophyll a/c-binding protein A (fcpA) terminator sequence, both of which were searched from the whole genome database using the Blast algorithm, were synthesized with flanking SalI-EcoRI and PstI-SphI restriction sites at both ends, respectively (Integrated DNA technologies, Inc., Coraville, IA, USA). After both fragments were cloned into the SalI-EcoRI and PstI-SphI sites of pSP-NPT/H4, the artificially synthesized genes encoding an enhanced green fluorescence protein (GFP) or putative oil body-associated protein-GFP fusion proteins (Takara Bio Inc., Tokyo, Japan) were inserted into the EcoRI-PstI sites between the promoter and the terminator. The transformation of strain JPCC DA0580 was performed by microparticle bombardment31-33 using the Biolistic PDS-1000/He Particle Delivery System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). In brief, expression vectors (5 µg) were attached onto tungsten particles (3 mg, 0.6 µm in diameter, Kojundo Chemical Laboratory Co., Ltd., Saitama, Japan), according to the manufacturer's protocol. Approximately 5×107 algal cells were spread onto 1% agar (Ina Food Industry Co., Ltd., Nagano, Japan) plates (3 cm in diameter) containing f/2 medium. The cells were bombarded with 600 ng of plasmid DNA attached on the microparticles using a 1,100-psi rupture disc under negative pressure of 28 inches of mercury (inHg). After bombardment, the cells were incubated under 140 µmol·m-2·s-1 of constant illumination for 24 h. Then the cells were recovered from small agar plates using f/2 medium and re-spread onto 1 % agar plates (9 cm in diameter) containing f/2 medium and 500 µg·mL-1 G-418, (Roche Applied Science, Tokyo, Japan), which is an aminoglycoside antibiotic derived from neomycin, to select the 13



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transformants. Fluorescence microscopy After formation of colonies of the transformants, the individual colonies were transferred in 24-well plates containing f/2 medium with G418 (500 µg/mL) and incubated for 2 weeks. Resulting cells were observed using a fluorescence microscope (BX51; Olympus Corporation, Tokyo, Japan) equipped with a cooled digital camera (DP-70; Olympus), a NIBA filter set for GFP, and a WIG filter set for chlorophyll fluorescence. Bioinformatics Amino acid sequences of the identified proteins were submitted to BlastP, and were annotated when the e-values were less than 10-10. In case all proteins found by BlastP with the e-values less than 10-10 were hypothetical (or predicted) proteins, we described the protein function as “unknown”. In the case no protein was found by BlastP with the e-values less than 10-10, we described it as “no hit”. Subsequently, the proteins annotated as “unknown” were analyzed by InterProScan for domain search. Cellular localization of the identified proteins was predicted based on a series of bioinformatics tools; TargetP and HECTAR were used for predicting general protein localization. Signal peptides for endoplasmic reticulum- (ER-) or chloroplast-targeting peptides were screened using SignalP. When a cleavage site is predicted by SignalP, the presence of the ER retention signal (K(/D)-D(/E)-E-L) in the C-terminus was checked manually. If the proteins possessed no ER 14


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retention signals but contained F, W, Y or L at the +1 position of the cleavage sites, the proteins were considered to be chloroplast-targeted proteins. When proteins were predicted to be localized to the mitochondria by both TargetP and HECTAR, the proteins were considered to be transported into the mitochondria. In addition, Mitoprot was used to detect mitochondrial targeting peptides. If (i) the score of Mitoprot was > 0.9 or (ii) the score of Mitoprot was > 0.8 and mitochondrial localization was predicted by either of TargetP or HECTAR, the protein was also considered to be transported into the mitochondria. For proteins without a signal, chloroplast-targeting or mitochondrial-targeting peptides, peroxisomal-targeting signals at the C-termini (S(/A/C)-K(/R/H)-L(/M) or S-S-L) were checked manually. TMHMM was used to detect the presence of transmembrane regions. If transmembrane regions were predicted in the proteins without any targeting peptides, the possibility that the proteins might be localized to the ER could not be excluded. Proteins which do not have the characteristic peptide sequences described above were predicted to be localized in the cytoplasm. Hydropathy plots were generated employing the Kyte-Doolittle algorithm34, using the ProtScale program at http://www.expasy.ch/tools/protscale.html. The value G in each graph is the grand average of the hydropathy value (GRAVY) for each protein and was calculated by using the GRAVY calculator program at http://gravy.laborfrust.de/.

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Results Isolation of diatom oil bodies Because of the unique cellular structure of diatoms, in which ER membrane is considered to entirely cover both the oil bodies and chloroplasts, we predicted that isolation of the oil body-associated proteins from diatoms would not be as simple as the isolation from green algae, for which some successful examples have been reported35. Indeed, preliminary observation during disruption of the frustule of the Fistulifera sp. strain JPCC DA0580 revealed that chlorophyll molecules immediately dissolved into hydrophobic oil bodies (Fig. S1), strongly indicating the collapse of at least the chloroplasts; it follows that other components including proteins would also contaminate the oil body fraction. These facts guided us to decide that the oil body-associated proteins should be condensed by collecting oil bodies without completely excluding contamination from the chloroplasts and other fractions. Alternatively, oil body-specific proteins should be estimated by comparing the oil body fraction and other fractions afterward as mentioned in the discussion section. By BODIPY staining, it was confirmed that the two-phase incubation strategy used in this study could certainly give rise to intracellular oil accumulation (Fig. 1-A).

In order to isolate the oil

droplets as well as the associated proteins, we attempted two cell-disruption methods, beads beating and sonication. The beads beating process was capable of breaking the frustules effectively (Fig. S2) and subsequent ultracentrifugation step allowed oil droplets to form a solidified oil phase, the 16


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so-called lipid pad (Fig. 1-B), so that it could be readily separated from the water phase (Fig. 1-C). The lipid pad had green color indicating the contamination from chloroplasts as predicted. During this process, oil bodies embedded in the lipid pad were robust enough to sustain the original size and shape (Fig. 1-D), thus suggesting that the intact oil bodies were successfully condensed. In contrast, the sonication process was not suitable for oil body recovery because this method was so harsh that no oil fraction was observed because of intensive emulsification of the oil bodies, although cell breaking was successfully achieved as with the beads beating method (Fig. S2). Thus, further experiments employed beads beating as a homogenization method. For the evaluation of oil body recovery by the beads beating method, the oil content and associated proteins were quantified (Table 1 and 2). Approximately 58% of total oil was recovered from Fistulifera sp. strain JPCC DA0580 and 2.3% of total proteins were found to be included in the lipid pad. It should be noted that the sonication method could not recover oil bodies. Identification of the proteins extracted from the lipid pad The proteins recovered from the lipid pad and those dissolved in the water phase were applied to SDS-PAGE (Fig. 2). In both lanes, some bands were found at the same position, suggesting the presence of identical proteins. Afterward, all proteins in each lane were excised and processed for in-gel digestion, followed by nanoLC-MS analysis for identification. As a result, 41 proteins were identified from the oil body fraction, while 44 proteins were identified from the soluble fraction; 19 of the proteins were found in both fractions (Fig. 3-A and Table S1). 17



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The intracellular localization of the proteins identified from the lipid pad was predicted in silico and it confirmed the presence of a large number (twenty-eight) of cytoplasmic proteins (Fig. 3-B). These results indicated, as predicted, that a certain level of contamination of the oil body fraction by the other fractions was detected, and could not be prevented by our present techniques. In other words, the oil body-specific proteins could be determined by comparing the oil body and soluble fractions. As a result, we extracted 22 unique proteins from the oil body fractions (Fig. 3-A, Table 3). Fucoxanthin chlorophyll a/c protein could be caused by the fusion of chloroplast fraction as mentioned above. From the gel section including the glaring band around 17 kDa of oil body fraction, the peptides derived from fucoxanthin chlorophyll a/c binding protein (g7797) were identified (Inconsistency in size may be caused by the cleavage reaction during the cell disruption step, Fig. 2). Glutamyl endopeptidase, nuclear receptor co-repressor 1, Ulp1 protease family protein and mitochondrial/chloroplast ribosome small subunit component were reported to be localized to the nucleus or mitochondria in different organisms36, 37, and thus were removed from the list of oil body-associated candidates. Therefore, we tentatively determined that 15 proteins are oil body-associated protein candidates in Fistulifera sp. strain JPCC DA0580. To further observe in vivo localization, another narrowing down was attempted. Hydropathy plots were generated for the proteins identified in this study and compared with those for other oil body-associated proteins found in different organisms (Fig. S3). Significant hydrophobic cores, which were confirmed in oleosin and LDSP15, were not observed in this study. GRAVY indices of these proteins ranged from -0.83 to 0.24, 18


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showing slightly hydrophilic features but not uniform, which made it difficult to determine whether each protein associated with oil bodies. We therefore employed a more strict method, transmembrane domain prediction using SOSUI, for narrowing down to select the most likely proteins associated with oil bodies. A previous study on A. thaliana suggested that main oil body proteins tended to possess transmembrane domains, which could be for the association with oil droplets or their monolayer membrane38. Among the specific proteins in the oil body fraction, 5 proteins were predicted to be transmembrane proteins by the SOSUI program, and thus we subjected these proteins to further study in a GFP-fusion experiment. Localization study of the oil body-associated protein candidates To prove the in vivo localization of the tentative oil body-associated protein candidates, expression vectors for the five target proteins fused with GFP were constructed. When these vectors were introduced into this strain, transfomants were acquired in all cases, whereas fluorescence-emitting cells could be generated from only two vectors (for g4301-GFP and g6574-GFP). According to the BlastP analysis, g6574 was annotated as a potassium channel, NKT2-like protein. On the other hand the function of g4301 remained unknown based on BlastP, while domain search by InterProScan preliminary revealed that g4301 has a domain of alcohol dehydrogenase-like superfamily (Table 3). The identification of alcohol dehydrogenase-like superfamily was also found in the proteomics study of Camelina stavia oil body39. Fluorescence microscopy demonstrated that intense fluorescence was detected at the same positions with oil bodies in each transformant cell (Fig. 4), strongly supporting 19



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the localization of each fusion protein to the oil bodies. Furthermore, g6574-GFP, assigned as a potassium channel, was observed to be distributed at the space around the oil bodies too, which was assumed to be the ER membrane.

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Discussion In order to analyze oil body-associated proteins in microalgae, the cell disruption strategy is extremely important. In green algal cells, oil bodies and chloroplasts are structurally separated as different compartments. Therefore, though it is still technically difficult, some careful disruption methods worked to prevent chloroplast-contamination during oil body fractionation. For instance, Dunaliella cells were gently disrupted for this purpose by means of osmotic pressure so that intact organelles including oil bodies could be released. In the case of diatoms, a unique evolutional history through which primitive cyanobacteria were incorporated into heterotrophic eukaryotic cells (primary symbiosis) and further symbiosis occurred by incorporating the rhodophyta, formed by primary symbiosis, into other eukaryotic cells (secondary symbiosis), are considered to create highly-complicated membrane systems. Under electron microscopic observation, it was confirmed that chloroplasts were compartmentalized from the cytoplasm by four bilayer membranes, in which outer two membranes were directly connected to the ER membranes (the so-called ER-chloroplast membranes)35, 40 and oil bodies also closely interacted with them10, 11. Even in our strain, Fistulifera sp. strain JPCC DA0580, both chloroplasts and oil bodies looked to be inside the ER membranes. This complex of membranes acts as a barrier to purely separate oil bodies from other organelles. When the cells were gently broken under microscopy, quick fusion of chloroplast components into oil bodies was observed (Fig. S1), supporting the ease of contamination of oil bodies by other fractions. Therefore, we concluded that the purification of oil bodies from other compartments (e.g., 21



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chloroplasts, ER membrane and also soluble fraction) might be unreasonable by using our present techniques, and employed a different strategy, a subtraction method based on comparative proteomics analysis. In this method, diatom cells were homogenized by beads beating and then oil bodies (including oil droplets and associated proteins) were condensed by ultracentrifugation while contamination from other fractions was not strictly excluded. Another cell disruption method, sonication, failed to obtain enough proteins for further experiments because the oil droplets were fully emulsified, and the proteins associated with oil could not be collected even by ultracentrifugation. Therefore, we selected beads beating as the cell disruption method. Proteins included in the lipid pad were subjected to SDS-PAGE and nanoLC-MS/MS for protein identification. The soluble fraction after ultracentrifugation also underwent the same series of analyses. The target proteins that could specifically associate with oil bodies were estimated by subtracting proteins found in both fractions from all the proteins found in the lipid pad including contaminations. As a result, 22 proteins were discovered. Among these 22 members, some proteins that were presumed to be contaminants from the chloroplasts, mitochondria or nucleus were eliminated (three fucoxanthin chlorophyll a/c protein (g10448, g7797 and g18295), glutamyl endopeptidase (g5395), nuclear receptor co-repressor 1 (g8350), Ulp1 protease family protein (g17516) and mitochondrial/chloroplast ribosome small subunit component (g8627)). Contamination from these fractions also occurred in proteomics analysis in C. reinhardtii18, 41. At the end, 15 proteins were tentatively identified to be specifically 22


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associated to oil bodies in this study. Although the functions of 10 of these proteins were not known based on the amino acid sequences, some interesting features were found. Identification of SEC14-like cytosolic factor protein (g2311) might be consistent with the studies regarding fatty cells in animals where SEC14 was proposed to work as an intermediate to convey phosphatidylcholine between the ER and oil droplets 42. Five proteins among the specified 15 candidates were predicted to be transmembrane proteins by SOSUI program, suggesting more likely interaction with the monolayer membrane surrounding oil droplets. In accordance with this narrowing down, we attempted to reveal the localization of these 5 oil body-specific proteins by means of the GFP fusion technique. Transformants were obtained in all cases, but GFP fluorescence was detected only in two cases, g6574-GFP and g4301-GFP. This could be caused by the nontargeting recombination technique employed in this study, where the fusion gene had a chance to be inserted into the region rarely transcribed, and/or the inserted fragments could be truncated during recombination; further studies will be needed in the near future. In a g4301-GFP expression clone, green fluorescence was specifically confirmed only on the oil bodies. This protein has 506 amino acids in length and the function remained unclear. By contrast, fluorescence from g6574-GFP could be detected not only in oil bodies but in the ER. This protein has 524 amino acids in length, and was predicted to be a potassium channel, NKT2-like protein, thus suggesting an osmoregulation function at the ER. Overexpression of this protein was likely to distribute from the ER to oil bodies, providing indirect proof that the oil body monolayer membrane may originate from, or at least be connected to, the ER 23



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membrane. It should be noted that little chlorophyll signal from g6574-GFP expression clone could be attributable to the chloroplast shrink induced by intensive oil accumulation, which has been observed in this strain. As compared to other microalgae previously researched, an interesting feature of this study was the absence of major proteins like a major lipid droplet protein (MLDP) for C. reinhardtii and Dunaliella sp., lipid droplet surface protein (LDSP) for Nannochloropsis sp. and oil globule protein for H. pluvialis (Fig. 2). This observation was supported by the genomics analysis of this strain in which the ortholog genes of other oil body-associated proteins were not encoded. Repression of MLDP in C. reinhardtii by RNA interference resulted in the formation of larger oil bodies (~2.5 µm) than the controls (~1.5 µm), strongly suggesting that MLDP regulated oil body size18. Guided by these recent findings, the large size of oil bodies (~3.1 µm) observed in Fistulifera sp. strain JPCC DA0580 was postulated to be caused by the lack of size-regulation proteins like MLDP, although the results obtained in this study do not necessarily prove the absence of these major proteins in the oil bodies.

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Conclusions A proteomics approach was employed to identify the oil body-associated proteins in Fistulifera sp. strain JPCC DA0580. Through beads beating cell disruption and the subsequent ultracentrifugation, enough protein was obtained from the oil body fraction. This method did not aim to eliminate contamination from other fractions; such strict elimination has been considered to be necessary but has not yet been achieved. The contaminating factors could be removed by comparing proteins from the oil body fraction and the soluble fraction. This novel strategy successfully revealed fifteen proteins as oil body-associated protein candidates. Among them, two proteins, which were parts of proteins predicted to have transmembrane domains, were indeed confirmed to specifically localize to the oil bodies in this strain. This subtraction method worked certainly for the identification of the oil body-associated proteins, and will be applicable to other phases of cell growth or oil accumulation in order to elucidate the whole metabolism of TAG accumulated in oil bodies. This is the first report for proteins associated with diatom oil bodies to the best of our knowledge. Furthermore, distribution of such a protein implied that the origin of oil bodies might be derived from the ER membrane. The findings in this study could provide new insights into the elucidation of TAG biosynthesis, storage, and degradation in diatoms.

Acknowledgements This work was supported by JST, CREST. 25



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Supporting Information Available This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure legends Figure 1. Recovery of the oil body fraction from Fistulifera sp. strain JPCC DA0580. Oil accumulation in this strain was confirmed by differential interference contrast (DIC) observation and BODIPY505/515 staining (A, scale bar: 10 µm). Afterward, sucrose gradient ultracentrifugation allowed the separation of the lipid pad (B and C). The separated lipid pad contained the intact forms of oil bodies (D). Figure 2. SDS-PAGE analysis of the separated fractions from Fistulifera sp. strain JPCC DA0580. Lane 1: Soluble fraction, Lane 2: Oil body fraction, Lane M: Protein molecular weight marker. The sections excised from the gel separating the oil body-associated proteins and the soluble proteins are indicated on the right of each lane. Figure 3. Description of proteins identified from the oil body fraction and the soluble fraction separated from Fistulifera sp. strain JPCC DA0580 by nanoLC/MS analysis. (A) Comparative analyses of the identified proteins from each fraction. (B) Predicted localization of the proteins identified from the oil body fraction. Arabic numbers in the figures represent the numbers of proteins involved in each group. Figure 4. Microscopic observations for analysis of in vivo localization of recombinant proteins fused with GFP in Fistulifera sp. strain JPCC DA0580 transformants. The GFP fluorescence of g4301 and g6574 transformants was observed to be localized at the oil bodies. The predicted function for g4301 and g6574 are enclosed in parentheses. (scale bar, 10 µm) 27



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Table 1 Oil content of each fraction after sucrose gradient separation of Fistulifera sp. strain JPCC DA0580 cell disruptions. Oil contents were expressed with the unit of g/g of dry cell weight (dcw). Oil content [g/g-dcw] Bead beating

Sonication

Oil body fraction

0.30 (58 %)

N.D.

Soluble fraction

0.06 (12 %)

0.06 (12 %)

Pellet

0.16 (30 %)

0.47 (88 %)

Total oil

0.52

0.53

Table 2 Protein content of each fraction after sucrose gradient separation of Fistulifera sp. strain JPCC DA0580 cell disruptions. Protein contents were expressed with the unit of mg/g of dry cell weight (dcw). Protein content [mg/g-dcw] Bead beating

Sonication

Oil body fraction

3.4 (2.3 %)

N.D.

Soluble fraction

140 (96.2 %)

235 (96.3 %)

Pellet

2.2 (1.5 %)

9.0 (3.7 %)

Total proteins

146

244

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Table 3 Gene list of unique proteins in the oil body fraction of Fistulifera sp. strain JPCC DA0580. The genes shown in green color encode the proteins determined to be possible oil body-associated proteins with transmembrane regions in this study. The genes shown in gray color were considered as contaminations from the chloroplast, nucleus, and mitochondria.

Gene ID

Annotationa

kDa

Transmembrane regionb

GRAVY scorec

g4796

transmembrane protein

85

6

-0.11

g6705 g6574

ABC transporter transmembrane region potassium channel, NKT2-like protein

149 58

6 1

-0.33 -0.16

g4301 g5708 g10552 g19744 g1204 g5858 g11870

unknown unknown unknown unknown unknown unknown unknown

53 49 88 80 43 45 27

1 1 0 0 0 0 0

-0.030 -0.27 -0.71 -0.45 -0.77 -0.18 -0.35

g17204 g2223 g12717

unknown no hit unknown

105 88 40

0 0 0

-0.40 -0.45 -0.18

g2311 g10448 g7797 g18295

sec14-like cytosolic factor fucoxanthin chlorophyll a/c protein fucoxanthin chlorophyll a/c protein 2 fucoxanthin, chlorophyll protein 2

109 21 21 21

0 2 0 0

-0.076 0.19 0.24 0.17

g5395

glutamyl endopeptidase nuclear recepter co-repressor 1, isoform CRA_a Ulp1 protease family protein

47

0

-0.13

173

0

-0.83

134

0

-0.82

113

0

-0.50

65

0

-0.56

g8350 g17516 g2502 g8627

diatom spindle kinesin 1 Mitochondrial/chloroplast ribosomae small subunit component a

Annotated by BlastP (e-value < 10-10) b Predicted by SOSUI program c

Calculated by GRAVY calculator program

29



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a designated figure for abstract

33



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Figure 1 142x155mm (150 x 150 DPI)


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Figure 2 70x124mm (150 x 150 DPI)



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Figure 3 187x181mm (150 x 150 DPI)


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Figure 4 149x158mm (132 x 132 DPI)


Proteomics Analysis of Oil Body-Associated Proteins in the

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