Phosphorylation/dephosphorylation response to light stimuli of Symbiodinium proteins: specific light-induced dephosphorylation of an HSP-like 75 kDa protein from S. microadriaticum

Antibodies and reagents

Polyclonal anti-phosphothreonine (anti-pThr cat. 9381S) and anti- phosphoserine (anti-pSer cat. 2261S) antibodies were from Cell Signaling Technology™, Inc. (Danvers, MA). Monoclonal anti-phosphotyrosine antibodies (anti-pTyr sc-508) were from Santa Cruz Biotechnology (Dallas, TX). Monoclonal anti-actin antibody N350 was from Amersham; this antibody is commercially discontinued but we still maintain an available stock (Villanueva, Arzápalo-Castañeda & Castillo-Medina, 2014). Alkaline-phosphatase (AP) conjugated polyclonal anti-rabbit IgG and anti-mouse IgG antibodies raised in goat were from Zymed™ -Life Technologies (Grand Island, NY). The tripeptides RAD (Arg-Ala-Glu) o RGD (Arg-Gly-Glu) were from CALBIOCHEM®-Millipore (Billerica, MA, USA). Ethylene glycol-bis(2-aminoethylether)-NNN’N’-tetraacetic acid (EGTA), bovine casein hydrolyzate (N-Z-Amine A), and the amino acids, glutamic acid, arginine and glycine, were all from Sigma. Reagents 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) were from Promega (Madison, WI, USA).

Symbiodinium cell cultures

Dinoflagellate cultures of Symbiodinium kawagutii (referred as S. kawagutii) were a kind gift of Dr. Robert K. Trench (University of California at Santa Barbara). Symbiodinium sp. Mf11.5b.1 (referred as S. Mf11), and Symbiodinium microadriaticum Subsp. microadriaticum (also known as MAC-CassKB8 and from now on referred to as S. KB8) originally isolated from the jellyfish Cassiopea xamachana, were a kind gift of Dr. Mary Alice Coffroth (State University of New York at Buffalo). These cell lines correspond to clades F, B and A, respectively; they were routinely maintained in our laboratory in ASP-8A medium under photoperiod cycles of 12 h light/dark at 25 °C. Light intensity was maintained at 80–120 µmole quanta m⁻² s⁻¹.

Preparation of total protein extracts for phospho protein screening and responses to stimuli

Six-d-old Symbiodinium cells in ASP-8A culture medium were collected 4 h after the light phase of the photoperiod initiated, and were concentrated by centrifugation at 1,400×g for 8 min. The cell pellet was suspended with 500 µl of 1× Laemmli buffer (60 mM Tris-HCl, pH 6.8, 1% (w/v) sodium dodecyl sulfate (SDS), 10% (v/v) glycerol, 3% (v/v) β-mercaptoethanol, 0.02% bromophenol blue) (Laemmli, 1970), supplemented with 0.2 mM NaVO₃, 10 mM NaPPi and a cocktail of protease inhibitors (Complete®; Roche, Basel, Switzerland), and placed in screw-cap polypropylene vials with a 0.25 ml total volume of glass beads (465–600 µm diameter) previously cooled. Subsequently, the cells were lysed with vigorous shaking in a MINI-BEAD BEATER™ (Biospec products) at maximum speed for 3 min. Then, the cell lysate was heated at 95 °C for 5 min, and centrifuged at 16,000×g for 10 min. The supernatant was used for analysis in polyacrylamide gels by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and western blot.

Protein electrophoresis in SDS-PAGE gels and western blot

The protein extracts were separated in discontinuous denaturing gels (Laemmli, 1970), of 10% polyacrylamide in the separation zone [375 mM Tris-HCl, pH 8.8; 10% acrylamide/bis-acrylamide; 0.1% SDS; 0.1% ammonium persulfate (APS); 0.106% N, N, N, N’-tetramethylethylenediamine (TEMED)], and 4% polyacrylamide in the stacking zone [125 mM Tris-HCl, pH 6.8; 4% acrylamide; 0.137% bisacrylamide; 0.1% SDS; 0.1% APS; 0.066% TEMED], in a Mini-PROTEAN®3 System (Bio-Rad, Hercules, CA, USA). After electrophoresis, the proteins were transferred to PVDF membranes in “friendly buffer” (25 mM Tris-HCl, 192 mM glycine, 10% isopropanol, Villanueva, 2008) at a constant current of 300 mA for 1 h. The membranes were blocked in a solution of 3% bovine serum albumin (BSA) in PBS (2.79 mM NaH₂PO₄, 7.197 mM Na₂HPO₄, 136.9 mM NaCl, pH 7.5) for 1 h at 50 °C, with gentle agitation. After blocking, the primary antibodies, anti-pThr (1:2,500), anti-pSer (1:500), anti-pTyr (1:500) or anti-actin (1:1,000) diluted in PBS containing 0.01% Triton X-100 (PBS-T), were added to the membranes and incubated overnight with gentle rocking at room temperature. Then, the membranes were washed five times, 5 min each, in PBS-T, and incubated with the appropriate secondary antibodies (alkaline-phosphatase conjugated anti-rabbit IgG or anti-mouse IgG) at 1:2,500 dilution for 2 h at room temperature. Subsequently, the membranes were washed again five times, 5 min with PBS-T. Finally, they were developed with a commercial solution of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) according to the manufacturer (Promega, Madison, WI, USA) in PBS-T for the primary polyclonal antibodies to ensure more astringency and less background. In the case of primary monoclonal antibodies, the final wash was followed by a brief rinse with alkaline developing solution (100 mM Tris-HCl, 150 mM NaCl, 1 mM MgCl₂, pH 9), and development in the same buffer. Western blot incubations, washing and development were also carried out in PBS-T or TBS-T (20 mM Tris, 150 mM NaCl, 0.01% TX-100, pH 7.6) with identical results.

Two-dimensional gel electrophoresis and partial peptide sequencing

Six-d-old cell cultures of S. KB8 collected 2 h before the dark phase of the photoperiod, were concentrated by centrifugation and suspended in 50 ml of fresh ASP-8A medium. After a 12 h incubation in the dark, the cells were precipitated by centrifugation at 2,600×g, 5 min, 26 °C and suspended in 10 ml of 0.2 mM NaVO₃ and 10 mM NaPPi in PBS-T added with a Complete® (Roche, Basel, Switzerland) cocktail of protease inhibitors, taking special care to carry out the whole procedure under dark conditions. The cells were then disrupted with a French press (SLM-AMINCO French® pressure cell press) at a pressure of 20,000 psi. Then, the cell lysate was chilled on ice and centrifuged at 100,000×g, 1 h to recover the proteins from the supernatant.

Next, the protein pellet was dissolved with seven volumes of PB (2.79 mM NaH₂PO₄, 7.197 mM Na₂HPO₄, pH 7.6) and applied to a column with 1 ml matrix volume of DEAE-sephacel previously equilibrated with 50 ml 0.02 M NaCl in PB. Then, the column was washed with 10 ml of the same buffer and eluted with 2 ml of 0.4 M NaCl in PB. The eluted proteins were precipitated with 8 volumes of cold acetone (at −20 °C), and one volume of trichloroacetic acid (TCA) for 1 h at −20 °C. Then, the mixture was centrifuged at 5,000×g for 5 min and the protein pellet was washed twice with 2 ml of acetone at −20 °C. Afterwards, the proteins were dissolved in BioRad® ReadyPrep buffer (8 M urea, 2% CHAPS, 50 mM DTT, 0.2% w/v ampholytes 3/10 BioLite® from BioRad and bromophenol blue), and the solution was used to hydrate a 7 cm IPG strip (ReadyStrip pH 4-7; Bio-Rad, Hercules, CA, USA) for 12 h at 18 °C. Then, the proteins adsorbed in the strip were separated with a programmed current of 250 V, 20 min, 4,000 V, 2 h and 4,000 V to cover 10,000 V-h in a PROTEAN IEF cell (Bio-Rad, Hercules, CA). Subsequently, the proteins in the strips were first denatured in 1.5 ml of Buffer I® (Bio-Rad; 6 M urea, 2% SDS, 0.375 M Tris-HCl pH 8.8, 20% glycerol and 2% w/v DTT) for 15 min, and then in 1.5 ml of Buffer II® (Bio-Rad; 6 M urea, 2% SDS, 0.375 M Tris-HCl pH 8.8, 20% glycerol added with 2.5% w/v iodoacetamide) for 15 min. Then, the strip was briefly rinsed in running buffer (25 mM Tris, 192 mM Glycine, 0.1% SDS, pH 8.3.) and loaded onto a 10% polyacrylamide SDS-PAGE gel to separate the proteins in the second dimension. Finally, the proteins were either stained with colloidal coomassie blue, or transferred onto a PVDF membrane for analysis with anti-pThr antibodies. The gel spots corresponding to the anti-pThr immunodetected signal were excised with a pipet tip, placed in a sterile eppendorf tube with 50 µL milliQ water, and sent for partial peptide sequencing to the Unit of Proteomics of the Institute of Biotechnology from UNAM in Cuernavaca, Morelos, México. Each spot was also reanalyzed by western blot with the anti-pThr antibodies to ensure their identity. In parallel, an adjacent mock spot corresponding to a major Mr ∼77 kDa protein was included as negative control for the analysis. The most probable sites of phosphorylation in threonine from the sequences were predicted by the NetPhosK server (Blom et al., 2004).

Light treatments on Symbiodinium cells adapted to darkness

Six-d-old cultures from S. KB8, S. Mf11 and S. kawagutii from three biological replicates collected 2 h before the dark phase of the photoperiod, were concentrated and suspended in 40 ml of fresh ASP-8A medium to reach 0.8–1.4  × 10⁶ cells/ml. Equal aliquots of each phylotype were placed in four 15 ml Falcon tubes wrapped with aluminum foil. The cells in the tubes were allowed to adapt in the dark (12 h) during their night cycle. After this period, the cells from one of the tubes were concentrated and broken with glass beads without exposing them to light throughout the process (control), and the other three tubes were released from the aluminum foil to expose them to light. Protein extractions with glass beads were performed at 30, 60 and 240 min after light exposure, and the supernatant from the lysate was heated at 95 °C, 5 min, and used for the analysis. Equal loads of proteins were adjusted with equal aliquots of cells and also standardized by staining with coomassie blue in 10% polyacrylamide SDS-PAGE gels. Finally, the extracts were analyzed by western blot which included actin as an additional internal loading control. The bands from the triplicate samples were captured with a ChemiDoc-It2 Imager (UVP-Analytik Jena, Upland, CA, USA) and analyzed by densitometry with the system’s VisionWorks LS software, normalized with the internal actin control to obtain an arbitrary level of intensity for each antibody reaction. The results were integrated into graphs displaying the average band intensity of the three biological replicates for each treatment.

Treatments of Symbiodinium cells with various stimulating agents

Six-d-old S. KB8 cells from a 1 L culture were collected by centrifugation at 1,400×g for 8 min, and suspended in 10 ml fresh medium, they were then divided into 9 aliquots of 1 ml in 15 ml Falcon tubes. The cells were concentrated again by centrifugation as above and the pellets were resuspended in: (a) 10 ml of fresh ASP-8A medium (control); or (b) 10 ml fresh medium supplemented with either 0.1% (w/v) casein, 10 mM glycine, 10 mM arginine, 10 mM glutamic acid, 100 µg/ml of the tripeptides RAD (Arg-Ala-Glu), RGD (Arg-Gly-Glu) (CALBIOCHEM®; Millipore, Billerica, MA, USA), 15 mM EGTA (ethylene glycol-bis (2-aminoentylether)-NNN’N’-tetra acetic acid), or 20 mM CaCl₂. All samples were incubated for 30 min at room temperature and after the incubations, the cells were concentrated by centrifugation followed by protein extraction with glass beads using the Bead Beater. Equal loads of proteins were analyzed by protein staining on SDS-PAGE and western blot with anti-pThr antibodies.

Anti-pTyr, -pSer and -pThr antibodies immunodetect several phosphoproteins from S. kawagutii, S. KB8 and S. Mf11 total extracts

Protein extracts from the three phylotypes under study were analyzed with the three anti-phosphoaminoacid antibodies by western blot. Anti-pTyr antibodies revealed intense bands with Mr ∼32 kDa in all three species (asterisk in Fig. 1C, lanes 1–3, respectively, and Fig. S1C), whereas species-specific bands of Mr’s ∼43, 57, 62 and 77 kDa were observed for S. KB8 (Fig. 1C, lane 1, and Fig. S1C, lane 1); of Mr’s ∼30 and 54 kDa for S. Mf11 (Fig. 1C, lane 2, and Fig. S1C, lane 2); and of Mr’s ∼48, 51 and 79 kDa for S. kawagutii (Fig. 1C, lane 3, and Fig. S1C, lane 3), indicating that these bands corresponded to proteins phosphorylated on Tyr residues but were not ubiquitous, except for the Mr ∼32 kDa protein. Anti-pSer antibodies revealed intense bands with Mr ∼65 kDa in all three species (Fig. 1B, asterisk in lanes 1–3, and Fig. S1B, lanes 1–3), and a weaker Mr ∼35 kDa band also in the three species analyzed (asterisk in Fig. 1B, lanes 1–3, and Fig. S1B, lanes 1–3). A relatively high number of weaker bands both ubiquitous and species-specific, were also detected with these antibodies in the three species analyzed (Fig. 1B, lanes 1–3, and Fig. S1B, lanes 1–3). These results indicated that a considerable number of proteins from all three species were phosphorylated on Ser residues. Finally, anti-pThr antibodies revealed several phospho proteins that were present in all three species analyzed. Here, we observed weak bands of Mr’s ∼43, 46, 50 and 55 kDa (asterisks in Fig. 1C, lanes 1, 2 and 3), and two clear bands with Mr’s ∼75 (double asterisk in Fig. 1A, lanes 1–3, and Fig. S1A, lanes 1–3) and 91 kDa (asterisk in Fig. 1A, lanes 1–3, and Fig. S1A, lanes 1–3). Additionally, the anti-pThr antibodies detected a clear band of Mr ∼29 kDa present in S. KB8 and S. Mf11 (Fig. 1A, lanes 1 and 2, and Fig. S1A, lanes 1 and 2) as well as a clear Mr ∼31 kDa protein band present only in S. kawagutii (Fig. 1A, lane 3, and Fig. S1A, lane 3), and a Mr ∼107 kDa protein band present only in S. KB8 (Fig. 1A, lane 1, and Fig. S1A, lane 1). All the proteins found to cross-react with the anti-phospho amino acid antibodies and their corresponding Mr’s are shown in Fig. S1. These data indicated: (a) the presence of proteins also phosphorylated on Thr residues; and (b) that there were phosphorylated proteins on the amino acids Tyr, Ser and Thr, in all three Symbiodinium species. One protein of Mr ∼75 kDa, which at this point we named pp75, fulfilled our criteria of being present in all three species and presenting a clear reproducible reaction upon developing by western blot. Therefore, we chose to further analyze this protein for possible changes upon treatment of the cells with light/dark and other stimuli.

Some phosphorylated proteins in threonine responded to a light stimulus

Analysis by western blot with anti-pThr in cells from all three species adapted to darkness during the dark phase of the 12-h photoperiod showed that pp75 maintained a considerable degree of apparent phosphorylation under this condition (Figs. 2A–2C, lanes 1; arrow labeled 75). Conversely, when the cells were stimulated with light for 30 min and analyzed by western blot, the intensity of pp75 decreased significantly (Figs. 2A–2C, lanes 2; arrow labeled 75). The weak intensity of the band was maintained even after 60 and 240 min of exposure to light (Figs. 2A–2C, lanes 3 and 4, respectively; arrow labeled 75). These results indicated that this protein, during the dark phase, is significantly phosphorylated on Thr; however, it dephosphorylates after 30 min of a “shock” of light and remains dephosphorylated for at least 240 min of such light exposure. Interestingly, the opposite behavior was clearly observed for a Mr ∼43 kDa protein only in S. KB8 cells before the light stimulus. This protein was not observed as phosphorylated in Thr when the cells were adapted to darkness (Fig. 2A, lane 1; arrow labeled 43). However, after 30 min of light exposure, the anti-pThr antibodies detected an intense band, indicating an enhanced phosphorylated state (Fig. 2A, lane 2; arrow labeled 43), which was maintained even after 60 and 240 min of exposure to light (Fig. 2A, lanes 3 and 4, respectively; arrow labeled 43). Species-specific light-stimulated phosphorylation behaviors were observed for a number of proteins immunodetected with anti-pThr antibodies. One Mr ∼29 kDa protein with similar behavior was detected in S. KB8 and S. Mf11 (Fig. 2A and B, lanes 1–4; arrow labeled 29), two of Mr’s ∼40 and 107 kDa in S. KB8 (Fig. 2A, lanes 1–4; arrows labeled 40 and 107, respectively); and finally, one of Mr ∼31 kDa from S. kawagutii (Fig. 2C, lanes 1–4; arrow labeled 31). The dephosphorylation of pp75 on Thr was consistently and reproducibly observed in cells from all three species when the light stimulus was applied in comparison to the cells adapted to darkness before the stimulus. The revealed bands were specific and the whole membrane showed specific reactions of the 75 and 43 kDa bands upon development of the blot (Fig. 2). In both cases, we quantified the response by densitometric analysis of the blots using triplicate biological samples and the intensity of the bands was normalized to the reaction of an anti-actin monoclonal antibody as internal control. The results were normalized, averaged, subjected to ANOVA, and graphed. In all cases, a statistically significant decrease in the phosphorylation level of pp75 in at least one time point after the light treatment was detected for all three species analyzed (Fig. 3A; S. KB8, lane 3; S. Mf11, lanes 2 and 3; S. kw, lane 4). The same analysis was carried out on the 43 kDa protein with similar results showing the opposite behavior for S. KB8 but not for the other two species (Fig. 3B). Although we cannot assure that they are the same as the 43 kDa protein that responds to light in S. KB8, Mr ∼43 kDa proteins from S. Mf11 and S. kawagutii cells, also recognized by the anti-pThr antibodies did not change their level of apparent phosphorylation after equivalent times after the light stimulus (Figs. 2B and 2C, lanes 2–4; Fig. 3B, S. Mf11 and S. kw, respectively), compared to the control (cells adapted to darkness, Fig. 2B and C, lanes 1; Fig. 3B, S. Mf11 and S. kw, respectively). This indicated that the Mr ∼43 kDa protein may have a species-specific behavior and only pp75 fulfilled the criterion of ubiquity in at least all three Symbiodinium species analyzed.

Threonine-phosphorylated proteins from S. KB8 did not show differential phosphorylation in response to other stimuli

In addition to light, we sought to stimulate S. KB8 cells in culture in order to observe possible changes in phosphorylation levels of proteins and relate those changes with other metabolic or signal-transduction pathways. Nevertheless, no changes were detected upon any of the treatments. None of the nutritional stimuli provided by incubation with the amino acids: glycine, arginine, glutamate, or a casein hydrolyzate caused significant changes in the degree of apparent phosphorylation in Thr of any protein including pp75 (Fig. S3, lanes 2, 3, 4 and 5, respectively), compared to the control (Fig. S3, lane 1). In parallel, extracellular matrix stimuli were tested with the tripeptide RGD (Arg-Gly-Asp), which is an inhibitor of the adhesion of integrins to their receptors. These RGD tripeptides did not cause any change on the phosphorylation of the analyzed proteins in S. KB8 cells (Fig. S3, lane 6), compared to the control without stimulation (Fig. S3, lane 1). As expected, the non-active tripeptide control RAD (Arg-Ala-Asp), did not cause any significant effect either (Fig. S3, lane 7). Finally, no alterations were observed in the phosphorylation pattern when the CaCl₂ concentration was increased in the medium (Fig. S3, lane 8), or when calcium ions were sequestered with EGTA (Fig. S3, lane 9), compared to the control (Fig. S3, lane 1). These results support the role of specific pThr phosphorylation as part of a light-regulated response in Symbiodinium.

The threonine-phosphorylated 75 kDa protein from S. KB8 is a BiP-like protein of the HSP70 protein family

The light-responding pp75 from S. KB8 was enriched through anion exchange column chromatography on DEAE-sephacel and the eluted fraction precipitated with ice-cold acetone-TCA, followed by analysis and separation by two-dimensional gel electrophoresis as described in ‘Materials and Methods’. The western blot of the separated proteins immunodetected with anti-PThr antibodies identified two Mr ∼75 kDa phosho protein spots of pI’s ∼5.4 and 5.6 (Fig. 4A, spots 1 and 2, respectively), indicating the presence of at least two isoforms of pp75. Only a few spots corresponding to the Mr’s of the phospho proteins previously immunodetected by the anti-PThr antibodies from S. KB8 extracts (Fig. S1A, lane 1) were observed, whereas a complex mixture of proteins was observed in the equivalent coomassie blue-stained 2D gel (Fig. 4B, spots 1 and 2). In addition, the excised spots as well as an adjacent mock spot (Fig. 4B, M) corresponding to a major protein of similar Mr, were re-analyzed by western blot with anti-PThr antibodies and only the lanes corresponding to the phospho protein spots were immunostained (Fig. S4, lanes 1 and 2), while the mock spot showed no cross-reaction (Fig. S4, lane 3). Both spots were sent for a partial peptide sequencing service and the identity from either spot matched a BiP-like protein sequence belonging to the HSP70 protein family from the S. microadriaticum transcriptome with 100% identity (Fig. 4C, Spot 1 and 2 sequences in red; Accession No. OLP91134). It is important to note that the reported sequence displayed a putative molecular weight of >200 kDa for the translated product, while pp75 migrates at Mr ∼75 kDa. Therefore, we analyzed this sequence further by comparing the corresponding genomic and transcriptomic sequences. This comparison revealed the correct start ATG and stop codons that resulted in the correctly translated product of 75 kDa. This corrected sequence is shown in Fig. 4C and it was used for a BLAST analysis against the S. microadriaticum transcriptome, which yielded at least other four related sequences encoding HSP70-like proteins (with long enough sequence showing relevant domains; accession numbers OLQ05431, OLP79133, OLP80221 and OLP86850), and one likely related short sequence (Accession No. OLP81269). Three of these sequences, including pp75, corresponded to BiP-like proteins (Accession No’s. OLP91134, OLP86850 and OLP81269). The sequence also contained 6 potential phosphorylated threonine residues (Fig. 4C, green Thr residues). These results indicated that the Mr ∼75 kDa protein from S. KB8 displaying differential phosphorylation on Thr upon a light stimulus was inequivocally identified as a BiP- or HSP70-like protein. Therefore, we renamed the protein as SmicHSP75.