Castillo-Medina Raúl Eduardo, Islas-Flores Tania, Morales-Ruiz Estefanía, Villanueva Marco A
Unidad Académica de Sistemas Arrecifales, Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México-UNAM, Puerto Morelos, Quintana Roo, México.
PLoS One. 2023 Oct 20;18(10):e0293299. doi: 10.1371/journal.pone.0293299. eCollection 2023.
The coding and promoter region sequences from the BiP-like protein SBiP1 from Symbiodinium microadriaticum CassKB8 were obtained by PCR, sequenced and compared with annotated sequences. The nucleotides corresponding to the full sequence were correctly annotated and the main SBiP1 features determined at the nucleotide and amino acid level. The translated protein was organized into the typical domains of the BiP/HSP70 family including a signal peptide, a substrate- and a nucleotide-binding domain, and an ER localization sequence. Conserved motifs included a highly conserved Thr513 phosphorylation site and two ADP-ribosylation sites from eukaryotic BiP's. Molecular modeling showed the corresponding domain regions and main exposed post-translational target sites in its three-dimensional structure, which also closely matched Homo sapiens BiP further indicating that it indeed corresponds to a BiP/HSP70 family protein. The gene promoter region showed at least eight light regulation-related sequences consistent with the molecule being highly phosphorylated in Thr under dark conditions and dephosphorylated upon light stimuli. We tested light parameter variations that could modulate the light mediated phosphorylation effect and found that SBiP1 Thr dephosphorylation was only significantly detected after 15-30 min light stimulation. Such light-induced dephosphorylation was observed even when dichlorophenyl dimethyl urea, a photosynthesis inhibitor, was also present in the cells during the light stimulation. Dephosphorylation occurred indistinctly under red, yellow, blue or the full visible light spectra. In additon, it was observed at a light intensity of as low as 1 μmole photon m-2 s-1. Our results indicate that: a) SBiP1 is a chaperone belonging to the BiP/HSP70 family proteins; b) its light-modulated phosphorylation/dephosphorylation most likely functions as an activity switch for the chaperone; c) this light-induced modulation occurs relatively slow but is highly sensitive to the full spectrum of visible light; and d) the light induced Thr dephosphorylation is independent of photosynthetic activity in these cells.
通过聚合酶链反应（PCR）获得了来自微小亚得里亚海共生藻（Symbiodinium microadriaticum）CassKB8的BiP样蛋白SBiP1的编码区和启动子区序列，进行测序并与注释序列进行比较。对应于完整序列的核苷酸被正确注释，并在核苷酸和氨基酸水平确定了SBiP1的主要特征。翻译后的蛋白质被组织成BiP/HSP70家族的典型结构域，包括信号肽、底物结合结构域和核苷酸结合结构域，以及内质网定位序列。保守基序包括一个高度保守的苏氨酸513磷酸化位点和来自真核生物BiP的两个ADP-核糖基化位点。分子建模显示了其三维结构中相应的结构域区域和主要暴露于翻译后修饰的靶点，这也与人类BiP紧密匹配，进一步表明它确实对应于一种BiP/HSP70家族蛋白。基因启动子区显示至少八个与光调节相关的序列，这与该分子在黑暗条件下苏氨酸高度磷酸化以及在光刺激下脱磷酸化一致。我们测试了可能调节光介导的磷酸化作用的光参数变化，发现只有在15 - 30分钟的光刺激后才能显著检测到SBiP1苏氨酸的去磷酸化。即使在光刺激期间细胞中存在光合作用抑制剂二氯苯基二甲基脲，也观察到了这种光诱导的去磷酸化。在红色、黄色、蓝色或全可见光谱下均发生去磷酸化。此外，在低至1微摩尔光子·平方米-2·秒-1的光强度下也观察到了去磷酸化。我们的结果表明：a）SBiP1是一种属于BiP/HSP70家族蛋白的伴侣蛋白；b）其光调节的磷酸化/去磷酸化很可能作为伴侣蛋白的活性开关发挥作用；c）这种光诱导的调节发生相对较慢，但对全可见光谱高度敏感；d）光诱导的苏氨酸去磷酸化与这些细胞中的光合活性无关。
