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

Abstract

to improve the photosynthetic performance of c 3 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.Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the incorporation of CO 2 into ribulose 1,5-bisphosphate (RuBP), which is indispensable for carbohydrate production in plants. However, Rubisco also catalyzes the oxygenation reaction of RuBP. This reaction has been considered wasteful since extra energy is consumed to recover RuBP and CO 2 is partially lost during the process of photorespiration. To compensate for the promiscuous nature of Rubisco, photosynthetic organisms have evolved various CO 2 -concentrating mechanisms (CCMs) 1,2 . For instance, cyanobacterial CCMs possess inorganic carbon (Ci) uptake systems and the microcompartments, carboxysomes, containing Rubisco. To date, five types of active Ci uptake systems have been identified in cyanobacteria. BicA and SbtA are single subunit sodium-dependent bicarbonate transporters on the plasma membrane 3,4 . In contrast, BCT1 is an ATP-binding, cassette-type bicarbonate transporter. The multimeric BCT1 complex is composed of four different subunits 5 . It has been proposed that the installation of CCM to chloroplasts is a promising approach to improve photosynthesis in C 3 plants. According to a theoretical estimation, installing any one of the bicarbonate transporters, BicA, BCT1, or SbtA alone, to the chloroplast inner envelope membrane (IEM) may improve photosynthesis 6-8 .It has been shown that chimeric cyanobacterial bicarbonate transporters expressed in the nucleus can be targeted to the chloroplast IEM 9,10 . Rolland et al. used a membrane protein leader (MPL) sequence that was fused to the N-terminus of the bicarbonate transporter together with the transit peptide 9 , which...

Engineered Accumulation of Bicarbonate in Plant Chloroplasts: Known Knowns and Known Unknowns

“…Finally, any processes which ensure activation and energization of the transporter must be met (discussed above). Correct targeting of HCO 3 − transporters to the chloroplast IEM has been the subject of several reports in recent years (Atkinson et al, 2016;Rolland et al, 2016Rolland et al, , 2017Uehara et al, 2016Uehara et al, , 2020Nölke et al, 2019), however, correct localization, orientation, and activation of these proteins to ensure favorable function remain an engineering challenge.…”

“…Frontiers in Plant Science | www.frontiersin.org site after the cTP to enable removal by a heterologously expressed TEV protease (Uehara et al, 2016(Uehara et al, , 2020.…”

“…These engineered cTPs, such as RC2 and PC1 ( Shen et al, 2017 ; Yao et al, 2020 ) include about 20 residues from its native mature cargo (a spacer to allow translocating factors better access to the cTP) which are followed by a second SPP cleavage site (to allow removal of the additional 20 residues used as spacer). Another approach that has been specifically used for the HCO 3 − transporters SbtA and BicA included a TEV protease cleavage site after the cTP to enable removal by a heterologously expressed TEV protease ( Uehara et al, 2016 , 2020 ).…”

“…As shown for the secretory pathway in plant endoplasmic reticulum, membrane protein signal peptides may also play a role in the orientation of some proteins ( Wojcik and Kriechbaumer, 2021 ). Hence, it is reasonable to assume that correct targeting and orientation of membrane proteins in the chloroplast IEM are dependent on both the cargo protein and its targeting sequence ( Rolland et al, 2016 ; Uehara et al, 2016 , 2020 ). As a result, broad screening of targeting peptides for candidate cyanobacterial membrane protein cargos is likely required, both on a case-by-case basis and possibly between heterologous hosts.…”

“…However, hurdles remain in both understanding and constructing CCM components within eukaryotic organelles where system complexity confounds an already difficult engineering task. This is exemplified by reports of the successful expression of HCO 3 − transporters into C 3 chloroplasts, but their lack of function and/or incorrect targeting ( Pengelly et al, 2014 ; Atkinson et al, 2016 ; Rolland et al, 2016 ; Uehara et al, 2016 , 2020 ), or lack of functional characterization in planta ( Nölke et al, 2019 ), highlights the need to further understand the composite interactions of chloroplast protein targeting, membrane energization, and small molecule passage across the chloroplast envelope.…”