- The chloroplast molecular machine for protein translocation : the function of a single complex and lipid effect (2011)
- Protein translocation across the chloroplast membrane is mediated by molecular machinery composed of protein complexes termed the TOC/TIC (the outer/inner envelope chloroplasts translocases). This translocation process is regulated by metabolic energy in form of GTP and ATP and is influenced by the lipid composition of the membrane. The ability to study the function of a single complex “TOC” in vitro using purified protein or purified chloroplast outer envelope vesicles has been instrumental for our understanding of the mechanism underlying this process. Indeed, the TOC complex has been purified by previously established procedures. However its functional and structural analyses are impaired by the limited yield of purified protein. Therefore, protocols for native TOC complex purification are described here. The complex isolation is achieved by direct biochemical treatment of biological membrane hosting this complex or by tandem affinity purification of modified protein complex components from generated transgenic plants. Furthermore, in this thesis, radioactive based in vitro import assays are described, namely those that allow monitoring translocation activity across the outer envelope of chloroplast. Based on the analysis of knock-out plants and isolated complexes it was previously suggested that lipid dependence of protein translocation might exist. Thus, the question was raised whether the lipid composition of the membrane has a direct influence on the behavior and functionality of the TOC translocon, or whether additional components of the chloroplast membrane account for the observed effect in vivo. To answer this question, a technique for vesicle fusion was developed. The principal aim was to explore the effect of an exchange of the lipid environment surrounding the complex translocon. This method helped to demonstrate that the SQDG and PI act stimulatory on the translocation across the outer envelope of chloroplast, whereas DGDG exhibits an inhibitory effect on TOC complex functionality.