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Investigating Localization of Chimeric Transporter Proteins within Chloroplasts of Arabidopsis thaliana   

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Original research article

A brief version of this protocol appeared in:
Frontiers in Plant Science
2016

Abstract

In this protocol, we describe a method to design chimeric proteins for specific targeting to the inner envelope membrane (IEM) of Arabidopsis chloroplasts and the confirmation of their localization by biochemical analysis. Specific targeting to the chloroplast IEM can be achieved by fusing the protein of interest with a transit peptide and an IEM targeting signal. This protocol makes it possible to investigate the localization of chimeric proteins in chloroplasts using a small number of transgenic plants by using a modified method of chloroplast isolation and fractionation. IEM localization of chimeric proteins can be further assessed by trypsin digestion and alkaline extraction. Here, the localization of the chimeric bicarbonate transporter, designated as SbtAII, is detected by Western blotting using antibodies against Staphylococcal protein A. This protocol is adapted from Uehara et al., 2016.

Keywords: Alkaline, Arabidopsis, Chimeric bicarbonate transporter, Chloroplast isolation, Chloroplast fractionation, Trypsin

Background

It has been proposed that the integration of cyanobacterial CO2 concentration mechanisms into chloroplasts is a promising approach to improve photosynthesis in C3 plants. According to theoretical estimations, integration of BicA and SbtA into the chloroplast IEM improves photosynthetic CO2 fixation rates. We examined the integration of nuclear-encoded cyanobacterial bicarbonate transporters, BicA and SbtA, to the IEM of chloroplasts in Arabidopsis. Therefore, we developed a protocol to design chimeric constructs for specific targeting of the IEM and investigate the localization of chimeric proteins in chloroplasts.

Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Uehara, S., Ito-Inaba, Y. and Inaba, T. (2018). Investigating Localization of Chimeric Transporter Proteins within Chloroplasts of Arabidopsis thaliana. Bio-protocol 8(3): e2723. DOI: 10.21769/BioProtoc.2723.
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