Maturation of iron-sulfur containing subunits of respiratory complex I in plants.
Résumé
Complex I (CI) is a major protein complex in the oxidative phosphorylation system of most of the eukaryotic mitochondria, being essential for the electron transfer chain (ETC) and for H+ pumping into the intermembrane space of mitochondria that is required for the formation of ATP by the ATP synthase (complex V) (1). The electron transfer to coenzyme Q is possible due to the presence of 8 Fe-S clusters (two [2Fe-2S] and six [4Fe-4S] clusters) bound by 5 subunits (51 kDa, 75 kDa, 24 kDa, PSST and TYKY). It is thought that the Fe-S clusters and other cofactors like FMN, are first assembled in the subunits, and afterwards, the subunits are assembled stepwise in larger assembly intermediates which will form the whole holo-complex I (2,3). The synthesis of Fe-S clusters in mitochondria is carried out by the Iron-Sulfur Cluster (ISC) assembly machinery. A [2Fe-2S] cluster is first de novo assembled on the scaffold protein (ISU). It may be directly transferred to [2Fe-2S] cluster-containing subunits. Additional transfer and conversion steps and players are then needed for [4Fe-4S] cluster-containing subunits. However, the exact mechanisms by which these five CI subunits obtain their Fe-S clusters from the transfer proteins of the ISC machinery still need to be elucidated (4). To clarify these mechanisms, we have expressed and purified several recombinant proteins corresponding to Fe-S containing CI subunits. With the ones that are soluble and stable (24kDa, TYKY), in vitro assays are performed to characterize their biochemical and Fe-S cluster binding properties. The ultimate goal is to perform in vitro Fe-S cluster transfer experiments using transfer proteins as donors. In parallel, we have generated CRISPR/Cas and RNAi mutants of three A-type carrier proteins (ISCA1a/1b and ISCA2) as well as of IBA57.1, which are known to act together in the transfer of Fe-S clusters to target proteins in mitochondria. The aim is to address the extent to which these proteins are involved in the assembly and function of CI. 1. Soufari, H., Parrot, C., Kuhn, L., Waltz, F., and Hashem, Y. (2020). Specific features and assembly of the plant mitochondrial complex I revealed by cryo-EM. Nature Communications 11: 5195. 2. Ligas, J., Pineau, E., Bock, R., Huynen, M. A., and Meyer, E. H. (2019). The assembly pathway of complex I in Arabidopsis thaliana. The Plant Journal 97: 447–459. 3. López-López, A., Keech, O., and Rouhier, N. (2022). Maturation and Assembly of Iron-Sulfur Cluster-Containing Subunits in the Mitochondrial Complex I From Plants. Frontiers in Plant Science 13. 4. Przybyla-Toscano, J., Christ, L., Keech, O., and Rouhier, N. (2020). Iron-sulfur proteins in plant mitochondria: roles and maturation. Journal of Experimental Botany: 2014-2044.