L'effet protecteur des HDL sur les pathologies cardio-vasculaires est principalement attribué à leur rôle central dans le Transport Retour du Cholestérol (TRC). Ce processus assure l'efflux du cholestérol excédentaire des cellules périphériques vers le foie, au niveau duquel il est éliminé dans les sécrétions biliaires. Dans ce contexte, notre équipe a identifié à la surface des cellules hépatiques la présence d’un complexe enzymatique, très proche de l’ATP synthase mitochondriale, comme étant un récepteur de haute affinité pour l’apoA-I (protéine majoritaire des HDL). Cette ATP synthase de surface, également appelée ecto-F1-ATPase, joue un rôle clé dans l’endocytose hépatique des HDL. En effet, la liaison de l’apoA-I stimule l’activité ATPasique de l’enzyme, entrainant la production d’ADP extracellulaire puis l’activation spécifique du récepteur nucléotidique P2Y13, aboutissant in fine à l’endocytose des HDL. Ainsi, l’équipe a montré le rôle clé de la voie ecto-F1-ATPase/P2Y13 dans l’endocytose hépatique des HDL et par conséquent dans les effets protecteurs de ces derniers dans l’athérosclérose.Les travaux de thèse présentés ici visent à déterminer les mécanismes de régulation de cette ecto-F1-ATPase. Compte tenu de l’importance de la régulation des taux d’ADP et d’ATP extracellulaires dans l’endocytose des HDL, nous nous sommes intéressés dans un premier temps aux acteurs moléculaires qui pourraient réguler le métabolisme nucléotidique à la surface cellulaire. Nous avons mis en évidence la présence, à la surface des cellules HepG2, de l’adénine nucléotide translocase (ANT), une autre protéine classiquement localisée à la mitochondrie. Nous avons montré que l’ecto-ANT est impliquée dans la régulation des taux des nucléotides adényliques ADP et ATP extracellulaires et que son fonctionnement est lui-même dépendant du taux de ces derniers dans le milieu extracellulaire. / The cardioprotective effect of high-density lipoprotein cholesterol (HDL-C) is mostly attributed to their metabolic functions in reverse cholesterol transport (RCT), a process whereby excess cell cholesterol is taken up from peripheral cells and processed in HDL particles, and later delivered to the liver for further metabolism and bile excretion. ATP synthase, classically known to be located in the mitochondrial inner membrane, has been unexpectedly found expressed at the plasma membrane of hepatocytes, as a receptor for apoA-I, playing a role in HDL-cholesterol uptake. On hepatocytes, apoA-I binding to ecto-F1-ATPase stimulates extracellular ATP hydrolysis into ADP, which subsequently activates a P2Y13-mediated HDL endocytosis pathway. The strict dependence of HDL endocytosis on extracellular ADP level led us to study first, whether other plasma membrane proteins than ecto-F1-ATPase could regulate extracellular ADP level. We highlighted the presence on hepatocytes cell surface of Adenine Nucleotide Translocase (ANT), another transmembrane protein of the inner mitochondrial membrane. We showed that ecto-ANT activity could increase or reduce extracellular ADP level, depending on the extracellular ADP/ATP ratio. Furthermore, we demonstrated that pharmacological inhibition of ecto-ANT activity increased extracellular ADP level when ecto-F1-ATPase was activated by apoA-I. This increase in the bioavailability of extracellular ADP accordingly translated into an increase of HDL endocytosis in human hepatocytes. We then sought to explore the molecular mechanisms involved in targeting ecto-F1-ATPase to the plasma membrane. Indeed, F1-ATPase ectopic expression at the plasma membrane has been described on several cell types and has been related to several physiological and pathophysiological processes however, the pathway involved in its transport to the cell surface remains unknown.

Dans le but de mieux comprendre le mode d’action et de nous permettre de déterminer la cible de la tomatidine, nous avons dans un premier temps tenté de mieux circonscrire le spectre d’activité de la tomatidine. Grâce à ces travaux, nous sommes, en effet, maintenant en mesure de dire que la tomatidine possède une activité antibactérienne contre les espèces de la division des Firmicutes et plus précisément contre les bactéries de l’ordre des Bacillales dont font partie les genres Bacillus, Staphylococcus et Listeria. Nous avons également découvert, grâce à des expériences en collaboration avec le laboratoire d’Éric Marsault, qu’un analogue de la tomatidine (FC04-100) avait non seulement des propriétés similaires à la molécule naturelle, mais démontrait une activité par lui-même contre S. aureus à phénotype normal alors que la tomatidine possède uniquement une activité contre les « small colony variants ». De plus, alors que la tomatidine possède plutôt une activité bactériostatique contre la forme SCV de L. monocytogenes, le nouveau composé (FC04-100) démontre quant à lui, une forte activité bactéricide contre cette souche, tout comme contre la forme SCV des autres Bacillales. Parallèlement, et toujours dans le but de rechercher le mode d’action et la cible de la tomatidine, nous avons obtenu, par passages successifs dans un milieu avec antibiotiques, des mutants de S. aureus à phénotype normal et des SCV résistants à la tomatidine ou à la combinaison tomatidine et gentamicine. Après le séquençage de ces mutants, l’étude de la position de ces mutations, à l’aide de différents logiciels de bio-informatique, nous a permis d’émettre un modèle-hypothèse quant au mode d’action et à la cible de la tomatidine. Selon les résultats que nous avons à ce stade-ci, la cible de la tomatidine chez S. aureus serait la sous-unité c de l’ATP synthase. Cependant, son mode d’action serait également dépendant de la fonctionnalité de la chaine de transport des électrons et donc de la polarisation membranaire et de la production de ROS intracellulaire, ce qui expliquerait la différence d’activité entre les souches à phénotype normal et les SCV.

Hemocytes (blood cells) play an important role in the immune response in invertebrates, and thus the regulation of hemocyte homeostasis (hematopoiesis) is essential for the host survival against pathogens. Astakine 1, a homologue to vertebrate prokineticins, was first identified in the freshwater crayfish Pacifastacus leniusculus as a cytokine, and was found to be necessary for new hemocyte synthesis and release in vivo, and also to induce spreading and proliferation of Hematopoietic tissue cells (Hpt cells, precursor of hemocytes) in vitro. The work of this thesis is aimed to further our understanding of the molecular mechanisms involved in astakine 1 induced hematopoiesis. Crayfish transglutaminase (Tgase) has been identified in the hemocytes, and is essential for the coagulation reaction. Interestingly this enzyme is exceedingly abundant in the Hpt cells, and the spreading of Hpt cells induced by astakine 1 was accompanied by sequential loss of TGase activity from the surface of these cells. This loss of TGase activity may be an important effect of astakine 1, resulting in recruiting new hemocytes into the circulatory system. Although astakine 1 contain a prokineticin domain, it lacks the conserved N-terminal AVIT motif present in its vertebrate homologues. This motif is important for vertebrate prokineticins to interact with their receptors, indicating a different receptor interaction for crayfish astakine 1. Astakine 1 was indeed found to interact with a completely different receptor, the β-subunit of ATP synthase, on a portion of Hpt cells, and subsequently block its extracellular ATP formation. Surface ATP synthase has been reported on numerous mammalian cells, but now for the first time in an invertebrate. The activity of ATP synthase on the Hpt cells may be important for the survival and proliferation of Hpt cells, but the underlying mechanisms remain further study. With the finding of a second type of astakine in crayfish, invertebrate astakines can be divided into two groups: astakine 1 and astakine 2. The properties of astakine 2 are different from those of astakine 1 both in structure and function. In primary cell culture of Hpt cells, only astakine 1 can promote proliferation as well as differentiation into semigranular cells, whereas astakine 2 may play a potential role in the maturation of granular cells. Moreover, a novel cysteine rich protein, Pacifastacus hematopoiesis factor (PHF), was found to be one target gene of astakine 1 in Hpt cells. Down regulation of PHF results in increased apoptosis in Hpt cells in vitro, and in vivo silencing PHF leads to a severe loss of hemocytes in the animal. Therefore astakine 1 acquires the anti-apoptosis ability by inducing its downstream gene PHF in the Hpt cells. With its ability to promote the survival, proliferation and differentiation of Hpt cells, astakine 1 is proven to be an important hematopoietic growth factor.

O gênero Hypostomus (Siluriformes: Loricariidae) com cerca de 130 espécies nominais, se destaca como um dos mais diversos e amplamente distribuídos gêneros de peixes de água doce neotropical. Devido a sua ampla distribuição e alta diversidade, os conhecimentos taxonômicos, filogenéticos e biogeográficos para as espécies do gênero são ainda consideravelmente incompletos. Consequentemente, pouco se sabe sobre processos naturais envolvidos em diversificação e variação morfológica para o gênero. Hypostomus ancistroides é uma espécie descrita para a bacia do Alto Paraná, uma eco-região hidrográfica tradicionalmente reconhecida por seu endemismo ictiofaunístico, ocorrendo também na bacia costeira do rio Ribeira de Iguape. Esta espécie apresenta considerável variação morfológica, cariotípica e isoenzimática em suas diferentes populações, sugerindo a existência de um complexo de espécies. Sua ampla área de distribuição, somada aos novos conhecimentos sobre padrões biogeográficos para diversas espécies de peixes do Alto Paraná, reforça essa possibilidade. Entretanto, a variação encontrada na morfologia das populações de H. ancistroides é ampla e contínua, impedindo que se defina diferentes espécies através das abordagens taxonômicas clássicas em ictiologia. Assim, este trabalho se propõe a utilizar ferramentas da sistemática molecular, genética de populações e filogeografia para responder questões fundamentais sobre a evolução desse potencial complexo de espécies. Sequências nucleotídicas completas do marcador mitocondrial ATP sintase (subunidades 6 e 8; 842 pb) foram obtidas para diversas espécies de Hypostomus, incluindo 162 exemplares de H. ancistroides provenientes de doze localidades abrangendo toda a sua área de ocorrência, além de outros gêneros da família Loricariidae, utilizados como grupos externos. Análises filogenéticas de Máxima Verossimilhança, Máxima Parcimônia e Neighbor Joining resultaram em topologias essencialmente semelhantes, sustentando a monofiletismo da espécie, e apontando como seus parentes mais próximos espécies de bacias hidrográficas adjacentes ao Alto Paraná. Esses resultados mostram ainda a existência de quatro filogrupos distintos para a espécie, com áreas de distribuição parcialmente sobrepostas. Análises populacionais e filogeográficas incluiram comparação de distância genética P, estruturação populacional baseada em distribuição de haplótipos e índices de diversidade, testes de neutralidade, índice de fixação FST, análise de variância molecular (AMOVA), análise espacial de variância molecular (SAMOVA), construção de rede haplotípica de parcimônia, e análise de clados hierarquizados (NCPA). Os resultados mostram 48 haplótipos repartidos em doze populações bem estruturadas, com baixo ou nenhum fluxo gênico entre si. Eventos de expansão geográfica podem ser identificados ao longo da história demográfica, sugerindo que a estruturação encontrada atualmente reflete não só as características ecológicas da espécie, como também uma história de mudanças nas condições ambientais, eventualmente favoráveis a migração e dispersão. Contatos entre populações de diferentes bacias podem ser mais frequentes através de capturas de cabeceiras do que ao longo do corpo dos rios principais. A hipótese mais plausível para a presença da espécie na bacia do Ribeira é a de uma captura de cabeceira do alto rio Tietê. Apesar de ser formado por quatro filogrupos distintos, algumas linhagens derivadas de H. ancistroides apresentam sobreposição de suas áreas de distribuição. Esse contato secundário revelado apenas por um marcador de herança matrilineal impossibilita a delimitação de diferentes espécies correspondentes aos filogrupos, sob os paradigmas clássicos de especiação em peixes neotropicais. / The genus Hypostomus (Siluriformes: Loricariidae), comprising ca. 130 nominal species, is one of the most species-rich and widely distributed genera of neotropical freshwater fish. Because of its wide distribution and vast diversity, knowledge on the taxonomy, phylogenetic relationships and biogeography of Hypostomus is still severely incomplete. Consequently, little is known about the processes involved in the diversification and morphological variation for the genus. Hypostomus ancistroides is a species described from the upper Rio Paraná drainage, a freshwater ecoregion known for its ichthyological endemism, also occurring in the coastal basin of Rio Ribeira de Iguape. This species shows considerable morphological, karyotypic and isoenzimatic variation among different populations, suggesting the existence of a species complex. Its wide distribution area, coupled with recent understanding on biogeographic patterns of several fish species from the Upper Parana, reinforces that possibility. However, morphological variation in populations of H. ancistroides is wide and continuous, and does not allow recognition of potential different species by means of traditional taxonomic approaches. Thus, this paper uses tools from molecular systematics, population genetics, and phylogeography in order to answer major questions about the evolution of this potential species complex. Complete sequences of the mitochondrial marker ATP synthase (subunits 6 and 8; 842 bp) were obtained for several species of Hypostomus, including 162 specimens of H. ancistroides from twelve localities covering its entire area of distribution, plus other loricariid genera as outgroups. Phylogenetic analysis using methods of Maximum Likelihood, Maximum Parsimony, Neighbor Joining and Bayesian Inference resulted in mostly similar topologies, supporting the monophyly of the species, and showing as its closest relatives other species from river basins bordering the Upper Parana. Results also reveal four distinct phylogroups for the species, with partially overlapping distribution areas. Population and phylogeographic analysis included comparisons of genetic distance P, population structure based on the haplotype distribution and diversity indices, neutrality tests, fixation index FST, analysis of molecular variance (AMOVA), spatial analysis of molecular variance (SAMOVA), construction of a parsimony haplotype network, and nested clade phylogeographical analysis (NCPA). Results show 48 haplotypes distributed into twelve well-structured populations with little or no gene flow. Geographic range expansion events can be identified along the demographic history of H. ancistroides, suggesting that the structure found today reflects not only the ecology of the species, but also a history of changing environmental conditions that on occasion weree favorable for migration and dispersal. Contact between populations from differente basins may be more intense through headwater stream capture than through the river channel. The most supported hypothesis for the presence of the species in the Rio Ribeira basin is a headwater capture from the upper Rio Tiete. Although H. ancistroides is split into four distinct phylogroups, some derived lineages of the species have overlapping distribution ranges. Such secondary contact is revealed only by a matrilineal inheritance marker and does not allow the recognition of separate species for the different phylogroups under the current paradigm of speciation and species limits in Neotropical fishes.

Binding of inorganic phosphate (Pi) in ATP synthase catalytic sites is a crucial step for the synthesis of adenosine-5'-triphosphate (ATP). ATP is the fundamental means of cellular energy in almost every organism, and in order to gain insight into the regulation of ATP catalysis, critical amino acid residues responsible for binding Pi must be identified. Here, we investigate the role of highly conserved α-subunit VISIT-DG sequence residues αSer-347, αGly-351, and αThr-349 in Pi binding. Mutations αS347A/Q, αG351Q, αT349A/D/R, βR182A, and αT349R/βR182A were generated via site directed mutagenesis. Results from biochemical assays showed that αSer-347 is required for transition state stabilization and Pi binding whereas αGly-351 is only indirectly involved in Pi binding and most likely maintains structural integrity of the catalytic site. Results from preliminary experiments on αThr-349 mutants suggest that the residue may be involved in Pi binding; however, further investigation is required to fully test this hypothesis.

L'ATP synthase ou ATPase de type F, ancrée aux membranes internes des mitochondries, est un complexe macromoléculaire qui utilise le gradient électrochimique généré par l'oxydation de petites molécules (NADH2, FADH2) dans les différents complexes de la chaîne respiratoire pour former l'ATP, vecteur énergétique universel. Le gradient électrochimique ou pmf est transformé en une énergie mécanique qui se traduit par le mouvement du rotor de l'ATP synthase dans un sens horaire vu depuis la membrane. La rotation de la sous-unité déforme successivement les trois sites catalytiques et permet ainsi la synthèse d'ATP. Dans certains cas, comme ceux de l'anoxie ou de l'hypoxie, le gradient électrochimique peut s'effondrer et l'ATP synthase hydrolyse alors l'ATP. Pour éviter cette hydrolyse futile, un petit peptide nommé IF1, régulateur spécifique des ATP synthases mitochondriales, vient s'insérer entre les sous-unités d'une interface catalytique et bloque instantanément le fonctionnement de l'ATPase. Cette inhibition est réversible puisque le peptide se décroche lorsque la membrane interne mitochondriale se réénergise. Dans ce travail de thèse, nous nous sommes intéressés à caractériser le mécanisme d'inhibition de l'ATPase de S.cerevisiae par son peptide endogène IF1 en s'appuyant essentiellement sur les quelques données structurales qui ont été publiées sur le peptide et sur le complexe inhibé IF1-F1-ATPase de B.taurus. Constitué de 63 acides aminés chez S.cerevisiae et 84 acides aminés chez B.taurus, IF1 est majoritairement structuré en hélice α. Les études menées par Elena Cabezón ont montré qu'IF1 possédait différentes formes dont la prédominance et l'activité dépendait essentiellement du pH. Chez B.taurus, il existe une forme inhibitrice dimérique prédominante à pH inférieurs à 6,5 et une forme tétramérique dont nous connaissons la structure 3D qui est non inhibitrice et prépondérante à pH supérieurs à 6,5. Chez S.cerevisiae, il existe une forme monomérique inhibitrice prépondérante à pH supérieur à 6,5 et une forme dimérique prédominante à pH inférieurs à 6,5 et dont le caractère inhibiteur ou non n'a pas encore été déterminé. Sur la base de la structure 3D de l'IF1 bovin, nous avons voulu identifier les régions de dimérisation du peptide de levure en utilisant la technique de marquage de spin couplée à de la spectroscopie RPE. En plaçant des marqueurs de spin (MTSL) en partie médiane (E33C) ou en C-terminale (L54C), nous avons pu favoriser l'interface de dimérisation plutôt en partie médiane du peptide. Ce travail est encore au stade embryonnaire et ne nous permet pas, à ce jour, d'identifier la zone exacte de dimérisation. Dans un deuxième volet, nous avons voulu caractériser le mécanisme d'inhibition d'un point de vue dynamique et nous avons pu en préciser les différentes étapes : reconnaissance, verrouillage et stabilisation. Pour cela, nous avons associé la mutagenèse sur le peptide et sur l'enzyme aux cinétiques d'inhibition. Nous avons tout d'abord évalué le rôle de plusieurs résidus situés en Cterminal de la sous-unité β, dans la région de l'interface α/ β qui se referme sur le peptide IF1, dans la reconnaissance moléculaire spécifique d'IF1 par l'ATPase mitochondriale. Nous avons ensuite montré que la partie N-terminale d'IF1 joue un rôle mineur dans la reconnaissance moléculaire mais son enroulement autour de la sous-unité constitue un loquet important dans la stabilisation du complexe inhibé. Enfin, la fermeture de l'interface catalytique sur IF1 crée une zone de contact entre la "bosse" de la sous-unité γ et la partie C-terminale de la sous-unité α qui constitue la dernière clef de blocage du peptide au sein de la F1-ATPase. Ce dernier point de fermeture est le seul qui n'implique aucun résidu du peptide IF1.

<p>The aim of the present study was to investigate the biosynthesis of the ATP synthase in various tissues, and to test hypotheses about possible models of activation of several mitochondrial proteins, the ATP/ADP translocase and UCPs, that could utilize the proton gradient, thus bypassing the ATP synthase. </p><p>We have examined the role of the expression of the P1 isoform of the c-F_o subunit in the biogenesis of ATP synthase in brown adipose tissue. Our findings point to a role for the c-F_o subunit in defining the final content of the ATP synthase in brown adipose tissue.</p><p>We have analyzed sequences in the 3’UTR of the β subunit F₁-ATPase mRNA that are important for formation of RNA-protein complexes. We could detect protein complexes that bind to two different sequence regions of the 3’UTR, one being the poly(A) tail and an adjacent region), and the other being a sequence stretch at the 3’ end of the 3’UTR able to form a stem-loop structure, which is evolutionarily conserved throughout mammalian species. </p><p>We investigated a role of the ATP/ADP carrier (ANT) in fatty acid-induced uncoupling in brown-fat mitochondria. We conclude that the ANT cannot substitute for UCP1 in fatty acid uncoupling in brown-fat mitochondria from mice lacking UCP1. We propose that the two ANT isoforms mediate proton translocation under different conditions.</p><p>We have investigated a role of UCP1 in defence against oxidative stress. We found that products of oxidative stress (4-HNE) could neither reactivate purine nucleotide-inhibited UCP1, nor induce additional activation of innately active UCP1 in brown-fat mitochondria from UCP1(+/+) and UCP1(-/-) mice. We conclude that UCP1 is not involved in defence against oxidative stress. </p><p>We evaluated possible uncoupling activity of UCP3 in skeletal muscle from warm- and cold-acclimated UCP1(+/+) and UCP1(-/-) mice. We conclude that no evidence exists for a higher UCP3-mediated uncoupling activity; a high UCP3 content in cold-acclimated UCP1(-/-) mice could possibly be linked to improved fatty acid oxidative capacity.</p>

The aim of the present study was to investigate the biosynthesis of the ATP synthase in various tissues, and to test hypotheses about possible models of activation of several mitochondrial proteins, the ATP/ADP translocase and UCPs, that could utilize the proton gradient, thus bypassing the ATP synthase. We have examined the role of the expression of the P1 isoform of the c-Fo subunit in the biogenesis of ATP synthase in brown adipose tissue. Our findings point to a role for the c-Fo subunit in defining the final content of the ATP synthase in brown adipose tissue. We have analyzed sequences in the 3’UTR of the β subunit F1-ATPase mRNA that are important for formation of RNA-protein complexes. We could detect protein complexes that bind to two different sequence regions of the 3’UTR, one being the poly(A) tail and an adjacent region), and the other being a sequence stretch at the 3’ end of the 3’UTR able to form a stem-loop structure, which is evolutionarily conserved throughout mammalian species. We investigated a role of the ATP/ADP carrier (ANT) in fatty acid-induced uncoupling in brown-fat mitochondria. We conclude that the ANT cannot substitute for UCP1 in fatty acid uncoupling in brown-fat mitochondria from mice lacking UCP1. We propose that the two ANT isoforms mediate proton translocation under different conditions. We have investigated a role of UCP1 in defence against oxidative stress. We found that products of oxidative stress (4-HNE) could neither reactivate purine nucleotide-inhibited UCP1, nor induce additional activation of innately active UCP1 in brown-fat mitochondria from UCP1(+/+) and UCP1(-/-) mice. We conclude that UCP1 is not involved in defence against oxidative stress. We evaluated possible uncoupling activity of UCP3 in skeletal muscle from warm- and cold-acclimated UCP1(+/+) and UCP1(-/-) mice. We conclude that no evidence exists for a higher UCP3-mediated uncoupling activity; a high UCP3 content in cold-acclimated UCP1(-/-) mice could possibly be linked to improved fatty acid oxidative capacity.

Le TMC207 est un nouvel antituberculeux appartenant à la famille des diarylquinolines qui inhibe très efficacement l'ATP synthase des mycobactéries. Dans le but de cartographier les interactions entre le TMC207 et sa cible et de comprendre le mécanisme d'action exact de cette nouvelle drogue, nous avons sélectionné in vitro des mutants résistants au TMC207 à partir de plusieurs espèces mycobactériennes. Six mutations distinctes ont été identifiées dans l'anneau c de l'ATP synthase: D28G, D28A, L59V, E61D, A63P et I66M. L'effet de ces mutations dans la résistance a été évalué en mesurant le niveau de résistance conféré dans les clones résistants, ainsi que dans un système de complémentation chez M. smegmatis. Les résultats ont été interprétés grâce à la construction d'un modèle structural de l'anneau c, utilisé pour faire des expériences de docking avec le TMC207. Nos résultats montrent que les résidus substitués dans les clones résistants définissent une poche localisée entre deux sous-unités c adjacentes dans l'anneau, englobant le site de fixation du proton, qui permet la stabilisation du TMC207. La drogue bloque ainsi le transfert des protons et la synthèse d'ATP. Pour finir, nous avons mis au point l'expression et la purification de l'ATP synthase mycobactérienne afin d'initier l'étude structurale de ce macro-complexe en microscopie électronique et en cristallographie des protéines. Les résultats obtenus en microscopie électronique en coloration négative nous ont permis d'obtenir les premières images de l'ATP synthase de M. smegmatis

E. coli SecA is an essential component for protein translocaiton across membrane. SecA can be deleted from its N- and/or C-terminal ends without losing complementation activity. In this study, we determined the dispensity of both ends of SecA molecule. The minimal length at the SecA C-terminus is dependent on the length of the N-terminal region. SecA10-826 and SecA22-829 are the two minimal length SecAs. One more amino acid deleted at the C-terminal end completely abolished their complementation activity. A hydrophobic amino acid is required at the 826th amino acid in the minimal-length SecAs. Both SecA22-828 and SecA22-829 could form a dimer, and have decreased ATPase and protein translocation activities. The active truncated SecA mutants tended to have more soluble form than membrane-bound form, but were stably embedded in membrane. In contrast, the inactive truncated SecA mutants tended to have more membrane-bound form, but were not stable in membrane. Thus, the loss of complementation is not related to dimerization, ATPase and translocation activity but to certain extent related to their biased subcelluar localization and conformation in membrane. Isolated membranes of E coli strains were solubilized and fractionated by sucrose gradient fractionation. These membranes fractions were depleted of SecY and YidC, but contained SecD, SecF and GroEL. Proteoliposomes reconstituted from these fractionated membrane proteins were active in pOmpA translocation which required SecA and ATP. Membrane fractions from strain CK1801 in which the unc gene is deleted were reconstituted into liposomes and also showed translocation activities. Moreover, proteoliposomes reconstituted with Bacteriorodopsin alone were not active in translocation, while proteoliposomes reconstituted with Bacteriorodopsin and CK1801 membrane fractions showed elevated translocation efficiency. These data suggested that proton motive force is not obligatory for, but stimulatory to translocation of pOmA. Purified GroEL was reconstituted into lipsomes and the reconstituted proteoliposomes were active in pOmpA translocation although at lower efficiency. This translocation also required SecA and ATP. These results together suggested that translocation of pOmpA is active in the absence of SecY and YidC. In the absence of SecYEG, translocation of pOmpA requires SecA and ATP. GroEL, SecD and SecF may participate in the SecY-independent translocation.