Cellular plant homeostasis : functional investigation of membrane protein insertion
Authors:   , Spies Nicole 
Published: February 2025

The outer envelope membrane of endosymbiotically derived organelles, like mitochondria and chloroplasts, exhibits characteristic, membrane integral β-barrel proteins. In contrast to the well-described mitochondrial counterpart, the biogenesis pathway of chloroplastidic β-barrel proteins is less understood. The similarities of the mitochondrial Sam50 protein, which acts as a β-barrel insertase in the outer envelope membrane, to its chloroplastidic orthologue Toc75-V lead to the assumption that Toc75-V also has a β-barrel insertase function. Gross et al., 2021 proposed an involvement of Toc75-V in the chloroplastdic β-barrel insertion pathway by showing a direct interaction of Toc75-V and the putative β-barrel substrate OEP37 prior to its final membrane maturation. However, the exact function of Toc75-V is still unknown. Gross, 2019 identified a so far uncharacterized component of the Toc75-V / OEP37 complex, namely BOP14 (β-barrel type protein assembly machinery on the outer envelope of plastids) encoded by the gene At5g64816. BOP14 showed similarities with some structural elements of the mitochondrial small Tim chaperones which are involved in the guidance of β-barrel proteins through the mitochondrial intermembrane space (IMS) towards Sam50. In addition to these similarities, the interaction of BOP14 with the putative insertase Toc75-V lead to the hypothesis of an involvement of BOP14 in the chloroplastidic β-barrel membrane insertion pathway. The small Tim proteins exhibit two structurally relevant intramolecular disulfide bonds (DSBs) formed by their highly conserved CX3C motifs. The cysteines of the small Tim proteins are involved in distinct steps of their biogenesis. However, the formation of the DSBs is necessary for the redox dependent oligomerization into the functional hexameric small Tim complex. The purpose of this study was to characterize BOP14 in terms of a putative redox- or cysteine dependent biochemical behavior, complex formation, interaction partners and its function. To achieve this, an in planta expression system (Pea early browning virus based pCAPE system, Fan et al., 2011) for P. sativum seedlings was employed (bop14-OX, bop14CL-OX), to achieve chloroplasts with the in planta expressed BOP14-Flag and its cysteineless mutant (BOP14CL-Flag). Due to the homologous expression of P. sativum BOP14 proteins in peas, it will be further reffered as overexpression (OX). The chloroplasts of bop14-OX and bop14CL-OX peas were isolated, solubilized under distinct redox treatments and subjected to non reducing SDS-PAGE.
The impairment of the germination, growth and development of young bop14-OX plants, which was not observed in bop14CL-OX plants, indicates a disulfide dependent bop14-OX phenotype formation. However, the suppression of BOP14 in bop14-KD plants showed a weak and chlorotic phenotype in later developmental stages. These older developmental stages exhibited an active RNA interference (RNAi) based BOP14 Knockdown (KD) and an already active protein turn over of BOP14. Therefore, no new BOP14 was expressed due to the KD while the BOP14 protein turn over led to the degradation of the previous present BOP14. Furthermore, based on distinct experiments with isolated chloroplasts of bop14-OX and bop14CL-OX (protease treatment and fractionation assay (localization) and HDN-PAGE (complex formation)) it could be shown that all ten cysteines of BOP14-Flag are necessary for its processing in the chloroplastidic IMS and for the complex formation with Toc75-V. The analysis of redox-treated bop14-OX chloroplasts in the non reducing SDS-PAGE revealed at least one intramolecular disulfide and it also forms intermolecular DSBs to a homodimeric and homooligomeric BOP14-Flag. Furthermore, the BOP14-Flag / Toc75-V complex formation was dependent on the cysteines of BOP14-Flag and could be assigned to the environmental redox conditions. BOP14M1-M4-Flag proteins with individual mutated CX3C motifs tend to undergo non-natural, reduction-sensitive oligomerization and the data indicates that all CX3C motifs are important for the native structure of BOP14. Furthermore, the diminished BOP14M1-M4-Flag interactions with Toc75-V suggests that all CX3C motifs are required for the interaction of BOP14 with Toc75-V. Using the LC-MS/MS after BOP14-Flag Co-Immunoprecipitation (Co-IP), distinct interactions of BOP14-Flag could be determined indicating interactions of BOP14 with components of a putative disulfide relay system for DSB recycling in the chloroplastidic IMS. The LC-MS/MS analysis also showed an interaction of BOP14 with Toc75-V and the putative β-barrel substrates OEP21, OEP24 and OEP37. Additionally, the protein reduction of the two putative Toc75-V substrates – OEP24 and OEP37 – in bop14-KD plants after protein turnover of the previous present BOP14 further suggests an involvement of BOP14 in the chloroplastidic β-barrel biogenesis. However, the exact role or function of BOP14 in β-barrel biogenesis needs further investigations. This also applies for the structure relevant BOP14 DSBs, formed from the components of a so far unknown chloroplastidic disulfide relay system.

References

Fan, Y., Li, W., Wang, J., Liu, J., Yang, M., Xu, D., Wang, X. (2011). Efficient production of human acidic fibroblast growth factor in pea (Pisum sativum L.) plants by agroinfection of germinated seeds. BMC Biotechnology, 11, 45.

Gross, L. E. (2019). Toc75-V in β-barrel biogenesis. Goethe university, Frankfurt.