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 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.

Figure 1

Construct designs for the chimeric bicarbonate transporters and tobacco etch virus (TEV) protease. (A) Schematic diagram of the chimeric BicA and SbtA constructs used in this study. The protein A domain (pA) of the fusion constructs contains two IgG-binding domains from staphylococcal protein A. The human influenza hemagglutinin (HA) domain consists of the amino acids YPYDVPDYA. Both BicA and SbtA genes are derived from Synechocystis sp. PCC 6803. The K124 construct lacks the 6th transmembrane domain of Cor413im1. TP, the transit peptide of Cor413im1; TEV, TEV recognition sequence (ENLYFQG). (B) Schematic diagram of TEV protease. The TEV protease gene is derived from the tobacco etch virus. RBCS–TP represents the transit peptide of the small subunit of Ribluse-1,5-bisphosphate carboxylase/oxygenase (Rubisco). MBP, Maltose binding protein. (C) Prediction before and after transformation with the TEV protease construct. When the bicarbonate transporter chimeric protein was co-expressed with TEV protease, we predicted that the TEV recognition sequence is digested by TEV protease in the chloroplast.

Figure 2

Expression analysis of chimeric BicA (A) and SbtA (B) with or without TEV protease in transgenic Arabidopsis. Total protein extracts (20 μg) from the rosette leaves were resolved by 12% SDS-PAGE and probed with antibodies against protein A. The arrowheads indicate the BicA– or SbtA–Cor413im1–protein A chimeric proteins. The arrows indicate the Cor413im1–protein A chimeric protein lacking a bicarbonate transporter. The asterisks indicate nonspecific proteins detected by the antibodies.

Figure 3

Localization of the chimeric proteins in the chloroplasts. Isolated chloroplasts (Cp) were fractionated into stroma (Str), envelope (Env), and thylakoid (Thy) fractions. The protein ratio of Cp to Str to Env to Thy used in these analyses was consistently 3: 3 :1:1.5. Each fraction was resolved by either 12% or 5–20% SDS-PAGE and immunoblotted with antibodies against protein A (pA; Cor413im1– and K124–protein A chimeric proteins), LSU, Tic110, LHCP, HA (HA-tagged authentic BicA and SbtA proteins), or MBP (MBP-fused TEV protease).

Figure 4

Trypsin sensitivity of BicA (A) and SbtA (B) chimeric proteins in the intact chloroplasts. Chloroplasts, equivalent to 25 μg of chlorophyll, were treated with trypsin on ice for 30 min. The trypsin was inactivated and the intact chloroplasts were re-isolated, resolved by either 12% or 5–20% SDS-PAGE, and immunoblotted with the antibodies against protein A (pA). The protease sensitivities of the outer envelope membrane protein, Toc75, and the inner envelope membrane protein, Tic110, were included as a positive and negative control to confirm the validity of the experiments, respectively.

Figure 5

Detection and comparison of HA- and protein A-tagged proteins in the chloroplast envelope membranes. Envelope fractions (14 μg for panels A and B, and 20 μg for panels C and D) were resolved by 5–20% SDS-PAGE and immunoblotted with antibodies against HA (A,C) or protein A (pA; B,D). The arrowheads indicate the full-length BicA– or SbtA–Cor413im1–protein A chimeric proteins. The arrows indicate the HA-tagged BicA and SbtA proteins. The closed circles indicate the Cor413im1–protein A and K124–protein A chimeric proteins. The asterisks indicate nonspecific proteins and degradation products detected by the antibodies.

Figure 6

Proposed model for the complementation test of a mutant plant lacking an IEM protein using a cyanobacterial orthologue. A nuclear-encoded cyanobacterial orthologue carrying a transit peptide (TP) and IEM targeting signal (IEM signal) is expected to be targeted to the IEM of chloroplasts. Then, TEV protease should cleave the chimeric protein, allowing the accumulation of the cyanobacterial orthologue on the chloroplast IME.