Sec translocon is the major machinery for protein translocation in E.coli including SecYEG, SecA and other Sec proteins. It is generally assumed that during translocation process, SecYEG serves as a protein-conducting channel and transports the protein across membranes by using SecA ATPase as driving force. However, previous work suggested that protein translocation can occur without SecYEG. In order to understand the role of SecA in this SecYEG-independent process, we use voltage clamp recording as a tool to study the ionic activity of SecA-dependent protein-conducting channel. In a major deviation from the conventional view, we found that SecA alone is sufficient to promote the channel activity with liposomes made of E.coli phospholipids in both whole cell recording in the oocytes and in the single channel recording with patch clamp. The activity is strictly dependent on the presence of functional SecA, including those from different species of bacteria. However, this SecA-alone dependent channel activity is less efficient compared to the membranes containing SecYEG. Furthermore, the channel activity loses the signal peptide specificity. Addition of purified SecYEG restores the signal peptide specificity as well as the efficiency. This channel activity is more sensitive to SecA-specific inhibitors compared with membranes containing wild-type SecYEG but is less sensitive to membranes containing suppressor proteins. This is the first time it has been shown that SecA binds to lipid low-affinity site and functions as a protein-conducting channel. To further characterize the structural roles of SecA as the core of the channel, we use several SecA variants to reconstitute with liposomes to determine the domains involved in forming functional channels. Using deletion truncated domains of 901 residues SecA and liposomes in the oocytes recordings, we identify two critical SecA domains for the formation of pore channel activity: with phospholipids alone, and for interacting with SecYEG to gain higher activity. These data provide fundamental understanding for the SecA-dependent protein –conducting channels. Our findings also suggest the possible evolution process on the protein translocation pathways from prokaryotes through eukaryotes.

Translocons are dynamic protein complexes with the ability to respond to specific signals and to transport polypeptides between two distinct environments. The Sec-type translocons are examples of such machineries that can interconvert between a pore forming conformation that translocates proteins across the membrane, and a channel-like conformation that integrates proteins into the membrane by lateral opening. This thesis aims to identify the signals encoded in the amino acid sequence of the translocating polypeptides that trigger the translocon to release defined segments into the membrane. The selected systems are the SecYEG translocon and the TIM23 complex responsible for inserting proteins into the bacterial and the mitochondrial inner membrane, respectively. These two translocons have been challenged in vivo with designed polypeptide segments and their insertion efficiency into the membrane was measured. This allowed identification of the sequence requirements that govern SecYEG- and TIM23-mediated membrane integration. For these two systems, “biological” hydrophobicity scales have been determined, giving the contributions of each of the 20 amino acids to the overall free energy of insertion of a transmembrane segment into the membrane. A closer analysis of the mitochondrial system has made it possible to additionally investigate the process of membrane dislocation mediated by the m-AAA protease. The threshold hydrophobicity required for a transmembrane segment to remain in the mitochondrial inner membrane after TIM23-mediated integration depends on whether the segment will be further acted upon by the m-AAA protease. Finally, an experimental approach is presented to distinguish between different protein sorting pathways at the level of the TIM23 complex, i.e., conservative sorting vs. stop-transfer pathways. The results suggest a connection between the metabolic state of the cell and the import of proteins into the mitochondria. / <p>At the time of doctoral defence the following papers were unpublished and had a status as follows: Paper nr. 1: Manuscript; Paper nr. 4: Manuscript</p>

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La diffusion de neutrons à petits angles (SANS) est une méthode utilisée pour l'étude d'une large variété de particules en solution. Combinée à un marquage isotopique et à la variation de contraste, elle permet d'obtenir des informations structurales uniques sur des complexes biologiques impliquant plusieurs partenaires, la rendant particulièrement intéressante pour l'étude des mécanismes de translocation. Dans les trois grands domaines de la vie, jusqu'à 30% des protéines doivent être sécrétées hors de la cellule ou intégrées dans sa membrane. Ces mécanismes complexes impliquent deux grands chemins de translocation qui dépendent de multiples protéines cytoplasmiques et membranaires.L'Holotranslocon est un large complexe protéique membranaire composé de sept sous-unités : le translocon triméric SecYEG et les sous-unités accessoires SecD-SecF-YidC-YajC. Cet assemblage peut sécréter des protéines vers l'extérieur de la cellule et en intégrer dans la bicouche lipidique. Nous avons utilisé une sélection de méthodes biophysiques pour analyser différentes stratégies de préparation et la diffusion de neutrons pour analyser les caractéristiques structurales de ce complexe. Nous avons étudié l'efficacité des protocoles actuels pour la production d'un échantillon suffisamment concentré et homogène mais aussi la possibilité d'utiliser des Amphipols comme substitut aux détergents classiquement utilisés pour la purification de ce complexe. Nous avons également tenter de deutérer HTL pour étendre les possibilités offertes par le SANS dans ce contexte. Alors que les caractérisations biophysiques utilisées n'ont pas permis d'améliorer les méthodes de préparation actuelles, la diffusion de neutron nous a permis de confirmer la présence de lipides au centre de la structure. Nous avons assuré la fiabilité de cette stratégie pour étudier des complexes bien définis, formés par plusieurs composants. Nous proposons des méthodes alternatives, basées sur la fluorescence ou la génération de lipodisques, pour l'étude de ce système complexe.Nous avons également étudié la protéine tétramérique SecB, une chaperonne moléculaire qui est impliquée dans l'adressage des substrats de translocation aux translocons membranaires. Puisque SecB interagit avec des partenaires cytosoliques et ses propres substrats, nous avons utilisé la diffusion de neutrons ainsi que le marquage au deutérium pour analyser des complexes pertinents dans le processus de translocation et impliquant jusqu'à trois partenaires : SecB, un substrat déplié et l'ATPase SecA. Nous avons utilisé les valeurs de "contrast match point" mesurées pour obtenir des informations sur la stœchiométrie et l'affinité des différents assemblages. L'analyse des rayons de girations a permis de localiser les différents composants au sein de ces complexes. De plus, nous avons montré que SecB s'étend lorsqu'elle reconnait son substrat et nous proposons un modèle fonctionnel, basé sur nos données, pour l'adressage des protéines textit{via} le chemin post-traductionnel.Dans l'optique de diversifier les applications du SANS pour les systèmes biologiques, nous avons étendus les protocoles utilisés pour la production de protéines partiellement deutérées textit{in vivo} aux lipides et acides nucléiques bactériens. Les niveaux de deutération ont été analysés grâce à la diffusion de neutrons, spectrométrie de masse ou résonance magnétique nucléaire. Sur la base de ces données, nous avons extrapolé les niveaux de deutération et "contrast match point" pour chaque type de biomolécule afin de pouvoir prédire les conditions de cultures optimales requises pour atteindre un marquage spécifique. Nous avons, de plus, développé une nouvelle stratégie permettant de marquer sélectivement les protéines tout en conservant un marquage minimal des autres types de molécules. / Small Angle Neutron Scattering (SANS) is a method that is used for the study of a wide range of particles in solution. Combined with isotope labelling and contrast variation approaches, it allows the extraction of unique structural information on biological complexes involving multiple partner molecules, making it a method of interest for the study of protein translocation mechanisms. In all three kingdoms of life, up to 30% of all proteins need to be secreted out of the cell or integrated into its membrane. These complex mechanisms involve two pathways of translocation that rely on multiple cytoplasmic and membrane proteins.The Holotranslocon (HTL) is a large membrane protein complex composed of seven subunits: the core trimeric translocon SecYEG and the accessory subunits SecD-SecF-YidC-YajC. This assembly can secrete proteins out of the cell or integrate them into the lipid bilayer. In this project, a range of biophysical methods and sample preparation strategies have been used to analyse the structural features of this complex by SANS. The efficiency of the current protocols to produce a concentrated and homogeneous sample have been investigated, as well as the use of an amhpipol molecules as a substitute to classical detergents for the purification of this complex. The deuteration of HTL was attempted to expand the capabilities of SANS in this context. While the biophysical characterization methods we used did not allow us to further improve the current preparation protocols, a key result was the fact that SANS confirmed the presence of lipids at the centre of the structure and the reliability of this analysis method for the study well-defined multi-component complexes was tested. Alternative methods for the analyses of this complicated system are proposed, including fluorescence-based assays or lipodisc formation.The tetrameric protein SecB was also studied; SecB is a molecular chaperone that is involved in the delivery of translocation substrates to the translocon. Since SecB interacts with cytosolic partners and its own substrate, SANS and deuterium labelling was used to analyse translocation-relevant complexes involving up to three partners: SecB, an unfolded substrate and the SecA ATPase. The measured contrast match point was used to obtain information on the stoichiometry and affinity of the various assemblies, and the radii of gyration was used to localize the different components within the complexes. It has also been shown that SecB expands upon substrate binding; a new working model for the post-translational targeting pathway has been proposed, based on these data.As a corollary to the work described above, protocols for textit{in vivo} protein deuterium labelling to the deuteration of textit{E. coli} lipids and nucleic acids have been developed and it is hoped that these may be of general value in widening the scope of SANS applications for the study of biological systems. the These approaches were characterized and evaluated by SANS, nuclear magnetic resonance and mass spectrometry. Based on these data, the relevant deuteration levels and contrast match points were extracted for each class of biomolecule so that the optimal culture conditions can be used to achieve a specific level of deuteration. In addition, a new strategy has been developed that allows the selective labeling of proteins while keeping the labelling of other classes of molecules to a minimum within a single culture.

Outer membrane proteins (OMPs) maintain the viability of Gram-negative bacteria by functioning as receptors, transporters, ion channels, lipases, and porins. Folding and assembly of OMPs involves synchronized action of chaperones and multi-protein machineries which escort the highly hydrophobic polypeptides to their target outer membrane in a folding competent state. Previous studies have identified proteins and their involvement along the OMP biogenesis pathway. Yet, the mechanisms of action and the intriguing ability of all these molecular machines to work without the typical cellular energy source of ATP, but solely based on thermodynamic principles, are still not well understood. Here, we highlight how different single-molecule studies can shed additional light on the mechanisms and kinetics of OMP biogenesis.

We have developed a novel, sensitive and less time-consuming method to detect activity of the SecA-dependent protein-conducting channels. Nanogram levels of E. coli inverted membrane vesicles were injected into Xenopus oocytes, and ionic currents were recorded using the two-electrode voltage clamp. Currents were observed only in the presence of E. coli SecA in conjunction with E. coli membranes. The observed currents showed outward rectification in the presence of KCl as permeable ions and were significantly enhanced by coinjection with the precursor protein, proOmpA, or active LamB signal peptide. Channel activity was blockable with sodium azide or adenylyl 5’-(β, γ-methylene)-diphosphonate, a non-hydrolyzable ATP analog, both of which are known to inhibit SecA protein activity. Channel activity was also stimulated by oocyte endogenous precursor proteins, which could be inhibited by puromycin. In the presence of puromycin, exogenous proOmpA or LamB signal peptides, but not defective signal peptides, stimulated the ionic currents. We also measured SecA-dependent currents with membranes depleted of SecYEG. Wild-type LamB signal peptides, or precursor proteins stimulated ionic currents following a co-injection of SecYEG¯ membranes with puromycin. Excess exogenous SecA stimulated ionic currents through SecYEG¯ membranes. Similar activities of added SecA were observed with reconstituted membranes depleted of SecYEG. Currents through such SecYEG-depleted membranes were also stimulated by addition of defective LamB signal peptides and unfolded mature PhoA protein. In contrast, currents produced by the membranes containing wild-type SecYEG were not so stimulated, but ionic currents were stimulated through mutant strains, similar to PrlA (SecY) suppressors, e.g. PrlA4, or PrlA665 membranes, suggesting that the proofreading function of SecY was bypassed in these membranes. We have observed that azide can inhibit ionic currents when E. coli wild-type MC4100 membranes were injected with proOmpA or LamB signal peptides into Xenopus oocytes. However, such inhibition was lost when observed with oocyte-endogenous signal peptides in the absence of bacterial signal peptides. Moreover, azide did not show complete inhibition upon using SecYEG¯ membranes or SecYEG¯ reconstituted membranes plus excess SecA in the presence or absence of LamB signal peptides. Such conformational alterations reflect different sensitivity in response to azide during the opening of protein-conducting channels.

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