The “revolution” in the resolution of cryo-electron microscopy opens new avenues to observe membrane proteins (MPs) in native membranes (1). Our lab contributed in the cryo-electron microscopy analysis of MPs by developing alternative surfactant named amphipol and by the genetic improvement of bacterial expression hosts (2, 3). Here we propose to study membrane biogenesis in the “AtpF model”. The overproduction of the b subunit of the FoF1-ATP synthase, AtpF, induces massive intracellular membrane (ICM) proliferation in C43(DE3) host (4), and preliminary results indicate that the expression of AtpF in the chloroplast of C reinhardtii might also shape its thylakoids membrane organization. Depending of its motivation and competences, the FP-DYNAMO fellow will have the opportunity to develop its project in several directions: i tomography on both models, Chlamydomonas chloroplast is a perfect material to study the thylakoids architecture by cryo-microscopy; ii, omic approaches; iii, genetics, by selective disruption of candidate genes involved in membrane biogenesis; iv, biochemical and synthetic biology approaches.  Our long-term project is to contribute to elucidate basic molecular mechanism of membrane proliferation across evolution and to apply them for the construction of a synthetic membrane organelle (5).

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References

1. Le Bon C, Michon B, Popot J-L, Zoonens M. 2021. Amphipathic environments for determining the structure of membrane proteins by single-particle electron cryo-microscopy. Q Rev Biophys 54:e6.
2. Angius F, Ilioaia O, Amrani A, Suisse A, Rosset L, Legrand A, Abou-Hamdan A, Uzan M, Zito F, Miroux B. 2018. A novel regulation mechanism of the T7 RNA polymerase based expression system improves overproduction and folding of membrane proteins. Sci Rep 8:8572.
3. Dilworth MV, Piel MS, Bettaney KE, Ma P, Luo J, Sharples D, Poyner DR, Gross SR, Moncoq K, J F Henderson P, Miroux B, Bill RM. 2018. Microbial expression systems for membrane proteins. Methods San Diego Calif 147:3–39.
4. Carranza G, Angius F, Ilioaia O, Solgadi A, Miroux B, Arechaga I. 2017. Cardiolipin plays an essential role in the formation of intracellular membranes in Escherichia coli. Biochim Biophys Acta 1859:1124–1132.
5. Royes J, Ilioaia O, Lubart Q, Angius F, Dubacheva GV, Bally M, Miroux B, Tribet C. 2019. Bacteria-Based Production of Thiol-Clickable, Genetically Encoded Lipid Nanovesicles. Angew Chem Int Ed Engl 58:7395–7399.