The major chaperones identified in Escherichia coli that assist in protein folding include trigger factor (TF), DnaK/DnaJ/GrpE and GroEL/GroES systems. The main ATP-dependent proteases are ClpXP, ClpAP, HslUV, Lon, and FtsH. From detailed sequence analysis, we found that tig (gene for TF), clpX, and clpP genes co-localize next to each other in most examined bacteria. We hypothesized that TF and ClpXP are functionally associated. TF is a ribosome-associated folding chaperone whereas ClpXP is a degradation complex. ClpX serves as the regulatory ATPase that recognizes substrates, unfolds and translocates polypeptides into ClpP for degradation. I found that TF physically interacts with ClpX, and that they collaborate to enhance degradation of certain ClpXP substrates. It is estimated that TF enhances the degradation of about 2% of newly synthesized E. coli proteins. One of the ClpXP substrates with degradation enhanced by TF was λO, the λ phage replication protein. Furthermore, TF also enhanced the degradation of ribosome-stalled λO nascent chains. Experiments suggest that TF transfers ribosome-stalled λO to ClpX for degradation by ClpP, demonstrating the existence of co-translational protein degradation in E. coli. To understand ClpXP mechanism, we had previously proposed that the degraded peptides are released from ClpP through transient equatorial side pores. To further understand ClpP dynamics, we determined the structure of ClpP(Ala153Cys) in its oxidized state. The structure shows that each opposing pair of protomers is linked by a disulfide bond. Unexpectedly, this structure resembles the compact structures of Streptococcus pneumoniae, Mycobacterium tuberculosis, and Plasmodium falciparum ClpPs, rather than the extended states seen in previous E. coli ClpP structures. Normal mode analysis of ClpP structures suggested that the iii compact structure is a naturally sampled conformation of WT ClpP. My findings provide insights for understanding ClpP dynamics as well as reveal a novel association between ClpXP protease and TF folding chaperone.

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Le premier acide aminé incorporé dans une chaîne polypeptidique naissante est toujours la méthionine. On identifie donc toujours ce premier résidu à la méthionine N-terminale. Cependant, les deux tiers des protéines accumulées à l’état stationnaire ne présentent plus leur méthionine initiatrice. Cet enlèvement résulte essentiellement d’une maturation protéolytique affectant chaque protéine. Ainsi, l’Excision de la Méthionine N-terminale (NME) concerne la majorité des protéines et ce dès que les premiers résidus émergent du ribosome. Ce mécanisme est retrouvé dans tous les compartiments cellulaires où une synthèse protéique a lieu : le cytoplasme, les plastes et les mitochondries. Les enzymes responsables du clivage de la méthionine initiatrice sont les METhionine AminoPeptidases (METAPs) ; les METAPs sont conservées dans le Règne vivant. Des études fonctionnelles de délétions géniques ont montré le caractère létal du maintien de la première méthionine dans tous les organismes. Il y a plus de dix ans, les METAPs ont été identifiées comme étant la cible de composés naturels ayant des effets anticellulaires. Aujourd’hui un nombre croissant d’études rapportent que la NME est une cible prometteuse pour le traitement de nombreuses pathologies. Néanmoins, les bases moléculaires qui expliquent le caractère essentiel de la NME restent très peu comprises, en particulier dans le cytoplasme des eucaryotes supérieurs. Grâce à un système inductible permettant de moduler finement la NME cytoplasmique dans la plante modèle Arabidopsis thaliana et différentes approches incluant des analyses protéomiques et métabolomiques, j’ai pu étudier les événements moléculaires précoces associés à l’inhibition de la NME cytoplasmique. J’ai également caractérisé la contribution relative des deux types de METAP cytoplasmiques au processus. Dans ce contexte, j’ai pu démontrer chez A. thaliana que la NME cytoplasmique agit sur deux voies de signalisation fréquemment dérégulées lors de conditions pathologiques : le statut des composés thiolés et la protéolyse. La diminution de la NME cytoplasmique induit une protéolyse accrue principalement via une augmentation du nombre de protéines destinées à une dégradation rapide. Ainsi, l’activité de la NME, en modulant la sensibilité de nombreuses protéines à subir la protéolyse, est un élément fondamental de la régulation de la demi-vie protéique. Finalement, mes résultats simialires obtenus également chez les Archées, levures et les lignées de cellules humaines suggèrent l’existence d’un mécanisme ubiquitaire associé à la NME. / The first amino acid incorporated in nascent polypeptide chain is always methionine so called N-terminale methionine. However, in a given proteome, more than fifty percent of proteins have not this first methionine. Indeed, the early proteolytic event affecting a majority of proteins is N-terminal Methionine Excision (NME) as soon as few residues exit from the ribosome. Enzymes ensuring NME process are conserved along species. This mechanism takes place in all compartments where protein synthesis occurs including cytoplasm, plastids and mitochondria and the enzymes responsible of N-methionine excision are METhionine AminoPeptidases (METAP). Early functional studies of gene deletion has quickly showed that NME is an essential process. Ten years ago, METAPs have been identified as the molecular target of natural compounds with anticancer activities. Now, a growing number of studies suggest that NME is a promising target for treatment of various deseases. Nevertheless, molecular mechanisms making NME an essential process is poorly understood in particular in higher eukaryote cytoplasms.Using a dedicated inducible system in the model organism Arabidopsis thaliana and multiple approaches, including proteomics and metabolomics, I examined the earliest molecular events associated with the inhibition of this process and the contribution of both METAP to NME process. In this context, I demonstrated that cytoplasmic NME in A. thaliana orchestrates a cross-talk between two fundamental signaling pathways frequently deregulated in pathological conditions: thiol status and proteolysis. In these studies, we demonstrated that developmental defects induced by cytoplasmic NME inhibition are associated with an increase of the proteolytic activity due to an increase of the proteins available for rapid degradation. Thus, NME activity that modifies the availability of several proteins for degradation is an integral and fundamental element protein turnover regulation. Finally my preliminary results obtained in Archea, Fungi and human cells seem to suggest the existence of a ubiquitous mechanism associated with NME process.

In der Hefe Saccharomyces cerevisiae können Proteine entweder co- oder posttranslational durch die Membran des Endoplasmatischen Retikulum transportiert werden. Sie besitzen eine Signalsequenz, die sie zu einem hydrophilen Kanal in der Membran bringt, durch den der Transport erfolgt. Die zentrale Komponente des Translokationsapparates in der Membran ist der aus den Untereinheiten Sec61p, Sbh1p und Sss1p bestehende Sec61p-Komplex. Beim Proteintransport wirkt der Sec61p-Komplex zusammen mit anderen Faktoren: Im cotranslationalen Transport geht er eine feste Bindung mit Ribosomen ein; der posttranslationale Transport erfordert die Assoziation mit dem tetrameren Sec62/63p-Komplex unter Bildung des sogenannten Sec-Komplexes. In der vorliegenden Arbeit wurde die Struktur des Sec61p-Komplexes durch Elektronenmikroskopie analysiert. Er liegt in Detergenzlösung in ringförmigen Strukturen mit einem Durchmesser von ~82Å und einer zentralen Pore von ~21Å vor. Jeder Ring besteht aus drei oder vier heterotrimeren Sec61p-Komplexen. Die oligomeren Ringstrukturen des Sec61p-Komplexes entsprechen vermutlich proteinleitenden Kanälen der Membran des Endoplasmatischen Retikulum. In Membranen wird ihre Bildung durch die Bindung von Ribosomen oder die Interaktion mit dem Sec62/63p-Komplex induziert. Eine dreidimensionale Struktur, die durch Kryo-Elektronenmikroskopie erhalten wurde, zeigt, daß das Ribosom so an den Sec61p-Komplex bindet, daß der Tunnel im Ribosom, durch den die naszierende Polypeptidkette das Ribosom verläßt, genau in die zentrale Pore des Sec61p-Oligomers mündet. Es existiert also ein kontinuierlicher Kanal, der sich vom Peptidyltransferase-Zentrum im Ribosom durch die zentrale Pore des Sec61p-Oligomers erstreckt, durch den naszierende Polypeptidketten cotranslational direkt in das Lumen des Endoplasmatischen Retikulum transportiert werden könnten. In dieser Arbeit wurde ein dem Sec61p-Komplex verwandter heterotrimerer Komplex in der Membran des Endoplasmatischen Retikulum identifiziert, der aus den Untereinheiten Ssh1p, Sbh2p und Sss1p besteht. Sss1p ist beiden trimeren Komplexen gemein; Ssh1p und Sbh2p sind homolog zu Sec61p bzw. Sbh1p. Durch Deletion von Ssh1p und Sbh2p wurde gezeigt, daß der Ssh1p-Komplex wie der Sec61p-Komplex am Transport von Proteinen in das Endoplasmatische Retikulum beteiligt ist. Der Ssh1p-Komplex ist mit membrangebundenen Ribosomen assoziiert und bildet in Detergenzlösung oligomere Ringstrukturen, aber interagiert nicht mit dem Sec62/63p-Komplex. Wir postulieren daher, daß der Ssh1p-Komplex ausschließlich den cotranslationalen Transport von Proteinen vermittelt. Beim posttranslationalen Transport interagiert das vollständig synthetisierte Modellsubstrat Prepro-Alphafaktor mit vielen cytosolischen Proteinen. Die cytosolischen Chaperone Hsp70 und TRiC konnten als Interaktionspartner identifiziert werden. Bei der Bindung des Prepro-Alphafaktors an die Membran werden die cytosolischen Proteine freigesetzt. Wir verwendeten einen Photoquervernetzungsansatz, um zu untersuchen, wie die Signalsequenz des Prepro-Alphafaktors im Bindungsschritt durch den Sec-Komplex erkannt wird. Die Signalsequenz-bindungsstelle wird hauptsächlich von Sec61p gebildet und befindet sich an der Grenzfläche zur Lipiddoppelschicht. Die gebundene Signalsequenz ist in einer helikalen Struktur fixiert und wird auf gegenüberliegenden Seiten von den Transmembrandomänen 2 und 7 des Sec61p umgeben. Sec62p und Sec71p, zwei Untereinheiten des Sec62/63p-Komplexes, flankieren gemeinsam eine Seite der Signalsequenzhelix, befinden sich aber in größerer Entfernung zur Signalsequenz als Sec61p. Es wird ein Modell vorgeschlagen, das beschreibt, wie die Bindung der Signalsequenz den Translokationskanal für den Transport öffnen könnte. / Protein transport across the membrane of the endoplasmic reticulum occurs either co- or posttranslationally in the yeast Saccharomyces cerevisiae. In both cases, polypeptides are directed to a translocation apparatus in the membrane by virtue of their signal sequences and then transported across the lipid bilayer through a protein-conducting channel. The major component of the protein translocation apparatus in the membrane is the heterotrimeric Sec61p complex consisting of the subunits Sec61p, Sbh1p and Sss1p. During translocation the Sec61p complex associates with other factors: In the cotranslational mode it interacts with ribosomes, whereas in the posttranslational mode it associates with the tetrameric Sec63/62p complex to form the so-called Sec complex. Here, we have analyzed the structure of the Sec61p complex by electron microscopy. In detergent this complex forms ring-like structures with a diameter of about 82Å and a central pore of about 21Å. Each ring contains 3 or 4 heterotrimeric Sec61p complexes. In membranes the formation of ring structures of the Sec61p complex is induced by its association with ribosomes or the Sec62/63p complex. We propose that the ring-like Sec61p oligomers represent protein-conducting channels of the endoplasmic reticulum membrane. A 3-dimensional structure of the ribosome-Sec61p complex obtained by electron-cryo-microscopy and single particle reconstruction showed, that the central pore of the Sec61p oligomer aligns precisely with the exit of a tunnel traversing the large ribosomal subunit that forms the passageway for the nascent chain. Thus, in cotranslational translocation a continuous channel extending from the ribosome through the Sec61p oligomer could guide the nascent chain directly into the lumen of the endoplasmic reticulum. Furthermore, we have discovered a trimeric protein complex in the yeast endoplasmic reticulum membrane that is structurally related to the Sec61p complex. This so-called Ssh1p complex consists of Ssh1p, a distant relative of Sec61p, of Sbh2p, a homolog of the Sbh1p subunit of the Sec61p complex, and of Sss1p, a component common to both trimeric complexes. In contrast to Sec61p, Ssh1p is not essential for cell viability, but it is required for normal growth rates. Sbh1p and Sbh2p individually are also not essential for cell viability, but cells lacking both proteins are impaired in their growth at elevated temperature and accumulate precursors of secretory proteins in the cytosol. Like the Sec61p complex, the Ssh1p complex forms ring-like structures in detergent and interacts with membrane-bound ribosomes, but it does not associate with the Sec62/63p complex. We therefore postulate that the Ssh1p complex functions exclusively in the cotranslational pathway of protein translocation. In the posttranslational transport process the newly synthesized translocation substrate prepro-a-factor associates with a large number of cytosolic proteins including the chaperones Hsp70 and TRiC. Upon binding of prepro-a-factor to the Sec complex all cytosolic proteins are released. Using a photo-crosslinking approach and a unique mapping technique we have investigated, how the signal sequence of prepro-a-factor is recognized by the Sec complex during the binding step. The signal sequence contacts primarily the multispanning membrane protein Sec61p. The bound signal sequence adopts a helical structure that interacts on opposite sides with transmembrane domains 2 and 7 of Sec61p, respectively. Sec62p and Sec71p, two subunits of the Sec62/63p complex, contact one side of the signal sequence, but are further away than Sec61p. Our data show, that the signal sequence binding site is located at the interface of the protein channel and the lipid bilayer. We suggest that binding of the signal sequence could open the channel for polypeptide transport.

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