Classically considered as a soluble system of enzymes, glycolysis does not conform to the known function and subcellular microcompartmentation pattern. Certain glycolytic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) could be found at different cellular locations in animal cells, where it exhibited its non-glycolytic activities. Determination of the subcellular localization of two cytosolic GAPDH isoforms from Arabidopsis thaliana (GapC1 and GapC2), fused to Fluorescent Proteins (FP), was investigated in the transiently transformed mesophyll protoplasts, using confocal Laser Scanning Microscopy. Apart from its cytosolic distribution, the nuclear compartmentation of GapC:FP was observed in this study, as well as its punctuate or aggregate-like localization. Part of the GapC:FP foci were observed as mitochondria-associated. A further yeast two-hybrid screen with both GapC isoforms as baits allowed to identify the mitochondrial porin (VDAC3; At5g15090) as a protein-protein interaction partner. Further tests with one-on-one yeast two-hybrid and Bimolecular Fluorescence Complementation (BiFC) assays showed that the detected binding between plant VDAC3 and GapC in yeast cells was false positive. Interestingly, aldolase interacted with VDAC3, as well as with GapC in plant protoplasts, using the BiFC method. On the other hand, no such interaction could be detected in the one-on-one yeast two-hybrid assay. Thus, a model of indirect mitochondrial association of GapC via aldolase that binds directly to mitochondrial porin is proposed to occur only upon certain cellular conditions. Attempts to show the binding of Arabidopsis GAPDH to the actin cytoskeleton in vivo failed, whereas in vitro cosedimentation assays demonstrated that the fully active, recombinant glycolytic enzyme binds to rabbit F-actin. Moreover, is the presence of the GapC cofactor NAD and a reducing agent (DTT), the enzyme might exhibit an actin-bundling activity.

Understanding the internal organization of cells from the molecular up to organelle level is a current challenge for biology if we are to better comprehend the mechanisms by which cellular processes occur. A prevailing view of the cell interior is that biochemical reactions and molecular movements are dominated by random diffusion. However, in addition to being compartmented into organelles, cells may well be organized at a finer level. Proteins may localise to specific areas within sub-cellular compartments, by associating with each other, the cytoskeleton and organelle membranes. Thus giving rise to distinct microcompartments. Considerable in vitro evidence exists for such interactions between enzymes and larger cellular components. This strengthens the idea that cells may be microcompartmented. However, little in vivo evidence supports this hypothesis, especially in plants. As a test-case for the concept of microcompartmentation this project investigated the sub-cellular distribution of the glycolytic enzyme fructose-bisphosphate aldolase in Arabidopsis thaliana; the ultimate aim being to establish whether it is microcompartmented in vivo and, further, to test the potential function(s) of such microcompartmentation.

En fournissant l’énergie nécessaire au métabolisme, la phosphorylation oxydative (OXPHOS) est un processus essentiel pour la plupart des organismes vivants. Pour faire face à diverses conditions métaboliques, l’efficacité des chaines respiratoires de la membrane composant l’OXPHOS doit être optimisée. Il est donc important de déterminer les mécanismes qui permettent de réguler l’efficacité de l’OXPHOS en fonction des besoins métaboliques.La question posée est la suivante : existe-t-il une organisation particulière des acteurs de l’OXPHOS dans la membrane des procaryotes qui puisse réguler l’activité de l’OXPHOS ?J’ai étudié l’organisation spatio-temporelle d‘un complexe respiratoire majeur de l’anaérobiose, la nitrate réductase NarGHI chez E. coli. Après avoir créé les outils pour la visualisation de ce complexe dans la cellule, j’ai montré l’existence d’une microcompartimentation de NarGHI aux pôles de la cellule lors d’une respiration en anaérobiose par microscopie optique à fluorescence. Dans un deuxième temps, j’ai montré le caractère dynamique de cette localisation en fonction des conditions métaboliques. L’anaérobiose et la présence d’un ∆pH suffisant sont des éléments requis pour permettre ce niveau d’organisation. Enfin, j’ai démontré que la microcompartimentation de NarGHI aux pôles des cellules augmente le flux d’électrons et l’efficacité des chaines respiratoires associées à la respiration du nitrate.L’ensemble des travaux réalisés au cours de ma thèse permet une meilleure compréhension du processus de l’OXPHOS chez les procaryotes et donne une nouvelle vision des moyens employés pour optimiser l’OXPHOS en fonction des différentes conditions métaboliques. / By providing the energy for the cellular metabolism, oxidative phosphorylation (OXPHOS) is an essential process for most living organisms. In order to thrive, the efficiency of membrane respiratory chains which constitute the OXPHOS must be optimized. Thus it is important to address mechanisms by which the efficiency of the OXPHOS is regulated in response to varying metabolic needs.The question addressed during this PhD is the following: does it exist a specific organization of the OXPHOS components in prokaryotic membranes and does it contribute to the regulation of the OXPHOS process?I have investigated the spatio-temporal organization of a respiratory complex, the nitrate reductase NarGHI of the E. coli bacterium. After creating the tools needed to visualize submicrometrically this complex in the unique cell, I have shown the existence of a polar microcompartimentation during anaerobic respiration using fluorescence microscopy. I have demonstrated the dynamic subcellular organization of NarGHI in response to metabolic conditions. Anaerobiosis and a sufficient ∆pH are cues required to promote such cellular organization. Finally, I have demonstrated that polar microcompartimentation of the complex increases the electron flux and the efficiency of the associated respiratory chains.Overall, these results provide a novel view on OXPHOS in bacterial cells by demonstrating that spatio-temporal organization of a respiratory complex tunes the overall efficiency of the process in response to environmental cues.