CGI58 (Comparative Gene Identifier 58) is considered a key regulator of lipolysis since the disruption of CGI58 in Arabidopsis results in the hyper-accumulation of lipid droplets in vegetative tissues. However, the molecular mechanism underlying this process has not been well studied. To address this, Arabidopsis lines stably expressing RNAi against transcripts encoding one or both of the CGI58 isoforms were generated and, using RT-PCR, specific alterations in CGI58 gene expression were shown to be achieved. Additionally, split-ubiquitin two-hybrid and bimolecular fluorescence complementation assays indicated that CGI58 interacts, albeit weakly, with PXA1, a peroxisomal transport protein responsible for the uptake of various lipid metabolites. Finally, results from mutational analyses of PXA1 revealed that the protein’s peroxisomal membrane targeting information is located within its N-terminal 180 amino acids. Taken together, these results have important implications for understanding the coordinated roles of CGI-58 and PXA1 in regulating lipid homeostasis in plants.

The integrated Membrane Yeast Two-Hybrid technology (iMYTH) was employed to screen the ABCD family of ABC Transporters in S. cerevisiae. The two ABCD members, Pxa1p and Pxa2p were screened against yeast libraries which detected many interactors involved in expected biological processes in accordance with Pxa1p and Pxa2p’s annotated function (i.e. fatty acid metabolism), and unexpected new interactors that may elucidate a new role for the transporters (e.g. oxidative protection). Members of the glutaredoxins and thioredoxins, and associated proteins were found to interact with Pxa1p and Pxa2p. This may indicate that Pxa1p and Pxa2p also play a role in managing the redox environment, protecting against reactive oxygen species. The iMYTH technology was also used to show that Pxa2p is able to form homodimers, and that Pxa1p localization is directly or indirectly dependent on Pxa2p.

The integrated Membrane Yeast Two-Hybrid technology (iMYTH) was employed to screen the ABCD family of ABC Transporters in S. cerevisiae. The two ABCD members, Pxa1p and Pxa2p were screened against yeast libraries which detected many interactors involved in expected biological processes in accordance with Pxa1p and Pxa2p’s annotated function (i.e. fatty acid metabolism), and unexpected new interactors that may elucidate a new role for the transporters (e.g. oxidative protection). Members of the glutaredoxins and thioredoxins, and associated proteins were found to interact with Pxa1p and Pxa2p. This may indicate that Pxa1p and Pxa2p also play a role in managing the redox environment, protecting against reactive oxygen species. The iMYTH technology was also used to show that Pxa2p is able to form homodimers, and that Pxa1p localization is directly or indirectly dependent on Pxa2p.

Les levures Candida sont des pathogènes opportunistes émergents. Après phagocytose macrophagique, C. albicans reprogramme son métabolisme pour faire face à une carence carbonée et induit 2 voies métaboliques, le cycle du glyoxylate et la β-oxydation. Notre objectif est d’étudier le lien entre β-oxydation, capacité de résistance à la phagocytose et virulence dans notre modèle biologique C. lusitaniae. Chez les levures Ascomycètes la β-oxydation, essentielle pour dégrader les acides gras (AG), est présumée être exclusivement peroxysomale.Nous avons construit 3 mutants nuls chez C. lusitaniae : icl1Δ, fox2Δ et pxa1Δ, respectivement défectifs pour l’isocitrate lyase (enzyme clé du cycle du glyoxylate), pour la protéine multifonctionnelle de la β-oxydation et pour une protéine responsable de l’import peroxysomal des AG à longue chaîne. L’étude de l’assimilation des AG et du catabolisme du 14Calpha-palmitoyl-CoA a révélé que les acyl-CoA à longue chaîne étaient toujours dégradés chez fox2Δ. L’étude du catabolisme des AG dans les fractions peroxysomale et mitochondriale des souches sauvage et fox2Δ, l’immunolocalisation de la protéine Fox2p et la mesure de la respiration mitochondriale nous ont permis de montrer pour la première fois chez une levure Ascomycète l’existence d’une β-oxydation Fox2p-dépendante dans la mitochondrie. C’est aussi la première démonstration chez un organisme eucaryote de la double localisation peroxysomale et mitochondriale de Fox2p. L’invalidation des gènes FOX1 et FOX3 (acyl-CoA oxydase et kétoacyl-CoA thiolase) a confirmé pour la première fois chez les champignons l’existence d’une voie peroxysomale alternative de catabolisme des AG, Fox2p-indépendante / The Candida spp. are emerging opportunistic pathogens. Phagocytic cells are a primary line of defense against these opportunistic pathogens. Upon phagocytosis by macrophages, C. albicans reprograms its metabolism because genes involved in the peroxisomal metabolism, such as glyoxylic acid cycle and beta-oxidation pathway, are overexpressed. The objective of this study was to study the relation between fatty acid beta-oxidation, resistance to phagocytosis and virulence in the biological model Candida lusitaniae. In ascomycetous yeasts, the fatty acid β-oxidation is assumed to be exclusively located to peroxisomes.We constructed three null-mutants in C. lusitaniae: icl1Δ, fox2Δ et pxa1Δ, respectively lacking the isocitrate lyase (a key enzyme of the glyoxylate cycle), the multifunctional fatty acid beta-oxidation protein (essential in C. albicans to the β-oxidation pathway), and a protein involved in the peroxisomal import of long-chain fatty acids. The study of fatty acid assimilation and 14Calpha-palmitoyl-CoA catabolism revealed that long-chain fatty acids were still catabolized in fox2Δ. The observation of 14Calpha-palmitoyl-CoA catabolism in mitochondrial and peroxisomal fractions of wild-type and fox2Δ strains, the immunolocalization of Fox2p and mitochondrial respiration measurements yielded to the first demonstration in ascomycetous yeast of a mitochondrial Fox2p-dependent fatty acid β-oxidation pathway. We also demonstrated for the first time in Eucaryota that Fox2p co-localized in both peroxisomes and mitochondria. The invalidation of FOX1 and FOX3 genes (acyl-CoA oxidase and ketoacyl-CoA thiolase, respectively) confirmed for the first time in Fungi the existence of an alternative peroxisomal pathway for fatty acid catabolism, Fox2p-independently.