Title : Role of the N-region in targeting multi-transmembrane domain proteins to the chloroplast
Abstract:
In C3 plants, the oxygenase activity of RubisCO leading to photorespiration and productivity loss can be curbed by introducing cyanobacterial carbon concentrating mechanisms (CCMs). The strategy's first phase envisages the introduction of the bicarbonate transporters (SbtA and BicA) to the chloroplast inner envelope membrane (IEM) to turbo charge RubisCOs CO2 supply. Targeting BicA and SbtA to the chloroplast IEM needs suitable IEM targeting signals, which are poorly characterized at present. Multiple transmembrane domain (TMD) proteins require a non-cleavable targeting signal in addition to the transit peptide (TP). We have generated constructs with the SbtA/BicA gene fused to the TP and MPL of Arabidopsis IEM proteins to test this. We find that the chloroplast import and membrane integration of multi-transmembrane domain (TMD)-containing proteins requires an unfolded sequence stretch beyond the transit peptide cleavage site, called the N-region. It is unclear if the N-region acts as an unstructured spacer or if its sequence plays a role in multi-TMD protein targeting. In this study, we have attempted to understand the N-region's role in chloroplast targeting of multi-TMD proteins by employing combinations of transit peptides and N-regions from Arabidopsis chloroplast inner envelope proteins to direct the heterologous multi-TMD protein SbtA to the chloroplast. Protoplast transfection and confocal microscopy imaging assay were used to investigate the sub-cellular localization of the chimeric proteins. We find that the successful import of multi-TMD proteins requires compatibility between the transit peptide and the N-region. Potentially, the N-region's charge distribution contributes to determining its compatibility with the transit peptide. The import potential of a compatible transit peptide- N region pair is specific to the type of cargo TMDs. Furthermore, we find that the transit peptide-N region sequence context plays a role in determining the intra-organellar destination of the heterologous multi-TMD protein. In the absence of a strong IEM signal in the TMDs of heterologous proteins, specific motifs in the N-region, such as the IQLP motif, can direct the protein to the thylakoid membrane. This study uncovers the crucial role of the N-region sequence context in the chloroplast import of multi-TMD proteins and helps us frame guidelines for directing heterologous multi-TMD proteins to specific chloroplast membranes.
Audience Take Away Notes:
- This work elucidates the import potential of a compatible transit peptide- N region pair in targeting a heterologous protein to the chloroplast
- The audience can make an informed decision when they design a leader sequence for specific intracellular targeting
- This research also indicates an absolute context specificity of molecular targeting sequences in transgenic/basic research
- Absolutely, a comprehensive analysis of several permutations/combinations of N terminal regions and Transit peptides will provide a reference for further research
- In transgenic experiments involving organellar transformation, it is often a serious issue to target a heterologous multi transmembrane domain protein to a specific membrane. Hence this study would greatly help those types of research
