Mapping SBiP1 protein-protein interactions in Symbiodinium microadriaticum CassKB8 using the Yeast Two-Hybrid assay and structural prediction

Abstract

BiP chaperones are central regulators of protein folding, endoplasmic reticulum homeostasis, and stress response across eukaryotes, but their client networks remain largely uncharacterized in Symbiodiniaceae. Here, we performed an initial yeast two-hybrid screening using a spliced-leader-based cDNA library to explore the interactome and regulatory dynamics of the Symbiodiniaceae BiP homolog, SBiP1, from Symbiodinium microadriaticum CassKB8. Using this approach, we identified eight candidate interactors with functions in translation, redox balance, RNA processing, and photosynthesis. AlphaFold2 structural modeling and Foldseek similarity analysis supported the plausibility of these candidates as BiP clients, revealing shared structural features such as globular folds and exposed hydrophobic or basic surfaces. Notably, two of the eight candidates, POX18 and TARBP1 were recovered in multiple independent clones, and even unannotated candidates displayed BiP-compatible surfaces. Gene expression analysis by RT-qPCR revealed dynamic transcriptional regulation of SBiP1 , HSP70 , and POX18 over an 18-day growth time course. All three genes peaked between days 6 and 12, suggesting coordination with intrinsic cellular cycles. SBiP1 expression was not significantly affected by light availability while HSP70 showed a modest but statistically significant increase following cycloheximide treatment, indicating transcript stabilization. In contrast, SBiP1 and POX18 expressions under the latter condition remained stable, suggesting their regulation may occur post-transcriptionally. Together, our findings highlight SBiP1 as a central player in ER proteostasis, bridging multiple cellular pathways essential for stress resilience. This work provides the first structural and functional map of a BiP-centered interaction network in a photosynthetic dinoflagellate, contributing to our molecular understanding of stress adaptation in these microorganisms.