Installation of authentic BicA and SbtA proteins to the chloroplast envelope membrane is achieved by the proteolytic cleavage of chimeric proteins in Arabidopsis

Susumu Uehara,Ayane Sei,Yasuko Ito-Inaba,Misaki Sada,Takehito Inaba

doi:10.1038/s41598-020-59190-1

Susumu Uehara, Ayane Sei + Show 3 more

Open Access

https://doi.org/10.1038/s41598-020-59190-1

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Journal: Scientific Reports	Publication Date: Feb 11, 2020
Citations: 5	License type: open-access

Affiliation: University of Miyazaki

Abstract

To improve the photosynthetic performance of C3 plants, installing cyanobacterial bicarbonate transporters to the chloroplast inner envelope membrane (IEM) has been proposed for years. In our previous study, we successfully introduced chimeric cyanobacterial sodium-dependent bicarbonate transporters, BicA or SbtA, to the chloroplast IEM of Arabidopsis. However, the installation of authentic BicA and SbtA to the chloroplast IEM has not been achieved yet. In this study, we examined whether or not tobacco etch virus (TEV) protease targeted within chloroplasts can cleave chimeric proteins and produce authentic bicarbonate transporters. To this end, we constructed a TEV protease that carried the transit peptide and expressed it with chimeric BicA or SbtA proteins containing a TEV cleavage site in planta. Chimeric proteins were cleaved only when the TEV protease was co-expressed. The authentic forms of hemagglutinin-tagged BicA and SbtA were detected in the chloroplast IEM. In addition, cleavage of chimeric proteins at the TEV recognition site seemed to occur after the targeting of chimeric proteins to the chloroplast IEM. We conclude that the cleavage of chimeric proteins within chloroplasts is an efficient way to install authentic bicarbonate transporters to the chloroplast IEM. Furthermore, a similar approach can be applied to other bacterial plasma membrane proteins.

Highlights

It has been shown that chimeric cyanobacterial bicarbonate transporters expressed in the nucleus can be targeted to the chloroplast IEM9,10
We examined whether or not tobacco etch virus (TEV) protease targeted within chloroplasts can cleave chimeric proteins and produce authentic bicarbonate transporters
To install authentic bicarbonate transporters to the chloroplast inner envelope membrane (IEM), we made a series of chimeric constructs possessing TEV protease cleavage site between the bicarbonate transporter portion and the other portion (Fig. 1)

Summary

Introduction

It has been shown that chimeric cyanobacterial bicarbonate transporters expressed in the nucleus can be targeted to the chloroplast IEM9,10. We used the mature portion of an IEM protein, designated as Cor413im[1], which contained an IEM targeting signal to deliver chimeric bicarbonate transporters to the chloroplast IEM10–12. This strategy achieved the expression of chimeric www.nature.com/scientificreports bicarbonate transporters in stable transgenic plants. In an in vitro cleavage assay, TEV protease was shown to cleave the chimeric BicA bicarbonate transporter on the isolated chloroplast IEM10. Our results indicate that the in vivo cleavage of the chloroplast-targeted chimeric transporter proteins can serve as an effective way to introduce authentic transporter proteins derived from other organisms This synthetic biology approach can be used to accelerate the metabolic engineering of plants

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Conclusion