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

Abstract

Background Some genera of the family Symbiodiniaceae establish mutualistic endosymbioses with various marine invertebrates, with coral being the most important ecologically. Little is known about the biochemical communication of this association and the perception and translation of signals from the environment in the symbiont. However, specific phosphorylation/dephosphorylation processes are fundamental for the transmission of external signals to activate physiological responses. In this work, we searched phosphorylatable proteins in amino acids of Ser, Thr and Tyr from three species of the family Symbiodiniaceae, Symbiodinium kawagutii, Symbiodinium sp. Mf11 and Symbiodinium microadriaticum. Methods We used specific antibodies to the phosphorylated aminoacids pSer, pThr and pTyr to identify proteins harboring them in total extracts from three species of Symbiodinium in culture. Extractions were carried out on logarithmic phase growing cultures under a 12 h light/dark photoperiod. Various light/dark, nutritional and other stimuli were applied to the cultures prior to the extractions, and proteins were subjected to SDS-PAGE and western immunoblotting. Partial peptide sequencing was carried out by MALDI-TOF on specific protein spots separated by 2D electrophoresis. Results At 4 h of the light cycle, several Thr-phosphorylated proteins were consistently detected in the three species suggesting a genus-dependent expression; however, most Ser- and Tyr-phosphorylated proteins were species-specific. Analysis of protein extracts of S. microadriaticum cultures demonstrated that the level of phosphorylation of two Thr-phosphorylated proteins with molecular weights of 43 and 75 kDa, responded inversely to a light stimulus. The 43 kDa protein, originally weakly Thr-phosphorylated when the cells were previously adapted to their 12 h dark cycle, underwent an increase in Thr phosphorylation when stimulated for 30 min with light. On the other hand, the 75 kDa protein, which was significantly Thr-phosphorylated in the dark, underwent dephosphorylation in Thr after 30 min of the light stimulus. The phosphorylation response of the 43 kDa protein only occurred in S. microadriaticum, whereas the dephosphorylation of the 75 kDa protein occurred in the three species studied suggesting a general response. The 75 kDa protein was separated on 2D gels as two isoforms and the sequenced spots corresponded to a BiP-like protein of the HSP70 protein family. The presence of differential phosphorylations on these proteins after a light stimulus imply important light-regulated physiological processes in these organisms.

Light-stimulated dephosphorylation of the BiP-like protein, SmicHSP75 (SBiP1) from Symbiodinium microadriaticum is inhibited by elevated but not low temperature and suggests regulation of the chaperone function

“…Then, they were processed immediately for analysis by western blot with anti-PThr or anti-SBiP1 antibodies. From all the sequences identified in the S. microadriaticum genome only one matched 100% with the peptide sequences obtained from 2-D gel spots (Castillo-Medina et al, 2019). We identified a 95 amino acid stretch near the Cterminus from such sequence (Fig.…”

“…The dialyzed proteins were then precipitated with the Cleanup ReadyPrep ® 2D kit (BioRad), according to the manufacturer's methodology. The proteins were suspended and separated by isoelectric focusing as previously described (Castillo-Medina et al, 2019). After isoelectrofocusing, the strips were briefly rinsed in running buffer and ran in the second dimension on a 10% polyacrylamide SDS-PAGE gel (16 cm×16 cm).…”

“…All light stimulation treatments and subsequent analyses were carried out as three independent experiments and following the previously reported procedures (Castillo-Medina et al, 2019). Six-d-old cultures from S. microadriaticum, F. kawagutii or B. psygmophilum collected 2 h before the dark phase of the photoperiod, were concentrated and suspended in 40 ml of fresh ASP-8A medium to reach 5-9×10 5 cells/ml (2-3.6×10 7 cells total).…”

“…Then, the cells from each tube were sedimented by centrifugation as previously described (Castillo-Medina et al, 2019), suspended in Laemmli buffer (Laemmli, 1970), supplemented with 0.2 mM NaVO 3 , 10 mM NaPPi and a cocktail of protease inhibitors (Complete ® ; Roche, Basel, Switzerland), mixed with glass beads (425-600 μm diameter) and lysed with a MINI-BEAD BEATER-1 ® (Biospec Products). The lysate was heated at 95°C for 5 min and the supernatant was used for western blot analysis.…”

“…We previously documented that in S. microadriaticum, SmicHSP75 was significantly phosphorylated in Thr under darkness, but the phosphorylation levels decreased after a 30 min light stimulus (Castillo-Medina et al, 2019). Since the protein is a member of the HSP70 family and its dephosphorylation is stimulated by light, we had particular interest in exploring the effect of low and high temperature stress on the modulation of the Thr phosphorylation of this protein.…”