Mitochondrial morphogenesis is a key process of cell physiology. It is essential for the proper function of this double membrane-delimited organelle, as it ensures the packing of the inner membrane in a very ordered pattern called cristae. In yeast, the mitochondrial ATP synthase is able to form dimers that can assemble into oligomers. Two subunits (e and g) are involved in this supramolecular organization. Deletion of the genes encoding these subunits has no effect on the ATP synthase monomer assembly or activity and only affects its dimerization and oligomerization. Concomitantly, the absence of subunits e and g and thus, of ATP synthase supercomplexes, promotes the modification of mitochondrial ultrastructure suggesting that ATP synthase oligomerization is involved in cristae morphogenesis. We report here that in mammalian cells in culture, the shRNA-mediated down-regulation of subunits e and g affects the stability of ATP synthase and results in a 50% decrease of the available functional enzyme. Comparable to what was shown in yeast, when subunits e and g expression are repressed, ATP synthase dimers and oligomers are less abundant when assayed by native electrophoresis. Unexpectedly, mammalian ATP synthase dimerization/oligomerization impairment has functional consequences on the respiratory chain leading to a decrease in OXPHOS activity. Finally these structural and functional alterations of the ATP synthase have a strong impact on the organelle itself leading to the fission of the mitochondrial network and the disorganization of mitochondrial ultrastructure. Unlike what was shown in yeast, the impairment of the ATP synthase oligomerization process drastically affects mitochondrial ATP production. Thus we propose that mutations or deletions of genes encoding subunits e and g may have physiopathological implications.

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Rho proteins are down-regulated in vivo by specific GTPase activating proteins (RhoGAP). We have functionally studied three Saccharomyces cerevisiae putative RhoGAP. By first identifying Rho partners with a systematic two-hybrid approach and then using an in vitro assay, we have demonstrated that the Bag7 protein stimulated the GTPase activity of the Rho1 protein, Lrg1p acted on the Cdc42 and Rho2 GTPases and we showed that Rgd2p has a GAP activity on both Cdc42p and Rho5p. In addition, we brought the first evidence for the existence of a sixth functional Rho in yeast, the Cdc42/Rac-like GTPase Rho5. ß

Because Lactuca sativa L. is a plant widely used in ecotoxicological analyses, a study was developed to determine whether the fatty acid composition of lettuce leaves could be used as an additional biomarker of soil contamination by metals such as Pb, Cu, Zn, and Cd. Unlike seed germination or seedling growth, the fatty acid composition of lettuce leaves differed significantly between uncontaminated and field metal-contaminated soils. Hence, this lipid biomarker might provide an early indication of a plant's exposure to metals and the potential bioavailability of metals, and could facilitate or strengthen the diagnosis of soil contamination. Not only is the experimental protocol cheap, rapid, and easy, but the values of the lipid biomarker are highly reproducible when seedlings are grown at the same light intensity. In addition, the values of the biomarker did not vary greatly when 14- to 18-day-old plants were analyzed and when slight differences were introduced in the experimental conditions used to determine the leaf fatty acid composition.

Small hydrophobic membrane proteins or proteins with hydrophobic domains are often difficult to produce in bacteria. The cell-free expression system was found to be a very good alternative for the expression of small hydrophobic subunits of the yeast ATP-synthase, such as subunits e, g, k, i, f and the membrane domain of subunit 4, proteins that are suspected to play a role in the stability of ATP-synthase dimers. All of these proteins could be produced in milligrams amounts using the cell-free "precipitate mode" and were successfully solubilized in the presence of lysolipid 1-myristoyl-2-hydroxy-sn-glycero-3-phospho-1'-rac-glycerol. Purified proteins were also found suitable for structural investigations. An example is given with the NMR backbone assignment of the isotopically labeled subunit g. Protocols are also described for raising specific polyclonal antibodies against overexpressed cell-free proteins.

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