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La particule de reconnaissance du signal (signal recognition particle-SRP) et son récepteur (FtsY chez Escherichia coli) médiatise le processus simultané de traduction-ciblage de la protéine en dirigeant le complexe ribosome-nascent chain (RNCs) vers la membrane de destination. La reconnaissance par la SRP d'une charge RNC à transporter dépend de la présence de la partie N-terminale. L'assemblage de Ftsy au complexe RNC-PRS entraine plusieurs changements de configuration de SRP et de FtsY durant le cycle de direction. D'abord un stade « précoce » sans GTP est adopté. Celui-ci est stabilisé par le RNC. Ensuite une configuration « fermée » avec GTP est formée. Cette dernière peut s'activer pour hydrolyser GTP, elle entre alors dans sa configuration « active ». La succession de ces trois étapes conduit à la libération du complexe SRP-récepteur d'avec le ribosome et de sa protéine en cours de traduction, et leur mise à disposition au pore de la membrane. Dans ce projet, notre intérêt se limite à la traduction par le ribosome de la séquence signale EspP (RNCEspP). In vivo, EspP est une protéine dont le ciblage vers le récepteur membranaire se réalise après la traduction. Cependant il arrive que RNCEspP se lie au complexe SRP-FtsY, faisant échouer le ciblage. Nous avons étudié les bases structurales du rejet de RNCEspP par SRP et FtsY. Pour cela nous avons effectué la comparaison de la structure RNCEspP-SRP-FtsY obtenue par observation au cryo-microscope électronique avec d'autres complexes ribosome-SRP-récepteurs traduisant la charge FtsQ, qui est elle normalement ciblé par SRP. Nous avons cherché à observer la différence de structure entre les complexes SRP-FtsY dans les deux cas. Deux différences majeurs entre les complexes de ciblages contenants les séquences RNCFtsQ et RNCEspP ont été observés. Premièrement, dans le cas de la structure de RNCEspP le domaine M -Ffh est attaché à l'hélice 59 du ribosome, alors que celui-ci est détaché dans le cas de la structure de RNCFtsQ. Nous pensons que le domaine M empêche la libération de la séquence de signal, étape nécessaire à la réalisation du ciblage. Deuxièmement, dans le cas de la structure du complexe avec RNCEspP l'arrangement Ffh-FtsY avec le domaine NG était flexible. Ceci indiquerait que le complexe “précoce” formé sur RNCEspP est moins stable que celui formé sur RNCFtsQ. Une étude biochimique utilisant le transfert d'énergie via résonance fluorescente a corroboré ce résultat, montrant que FTS Y est lié avec une affinité moindre dans le cas du complexe précoce formé sur RNCEspP et que la reconfiguration au stade de complexe fermé est moins efficace. Une analyse biochimique plus poussée des variantes de la séquence de EspP montre que la partie N-Terminale de la séquence est la principale cause de rejet du cycle de ciblage via SRP.Dans un second projet, nous avons étudié la configuration “fermée” de SRP et ftsY en complexe avec une charge RNC stabilisée par un analogue non-hydrolysable de GTP (GMP-PCP). Pour franchir la barrière cinétique qui permet de passer du complexe précoce au complexe fermé, nous avons utilisé une version tronquée de FtsY, à laquelle la séquence terminale avait été amputée de tout le domaine acide (A-) ainsi que de la première hélice alpha du domaine NG. De plus, pour la formation du complexe, nous avons utilisé une construction contenant les 50 premiers acides aminés du leader peptidase (RNCLep50). En l'absence de nucléotides, notre reconstruction au cryo-EM a montré une configuration similaire à celle du stade précoce, dans laquelle Ftsy et Ffh- domaine NG, sont proche du tetraloop de la 4.5 S ARN. Une incubation avec GMP-PCP induit un détachement du domaine NG d'avec la queue du tetraloop. Il semblerait que les domaines NG soient flexibles dans l'état clos, et non attaché à la terminaison ouverte de l'ARN. / The signal recognition particle (SRP) and its receptor (FtsY in Escherichia coli) mediate co-translational protein targeting by delivering ribosome nascent chain complexes (RNCs) to the target membrane. Recognition of an RNC cargo by SRP is dependent on an N-terminal signal sequence. Binding of FtsY to the RNC-SRP complex leads to several conformational changes of SRP and FtsY during the targeting cycle: first, an “early” GTP-independent state is adopted which is stabilized by the RNC, subsequently a “closed” GTP- dependent conformation is formed which can activate itself to hydrolyze GTP (the “activated” state). Faithful completion of all three steps leads to release of the cargo from SRP-FtsY and hand over of the RNC to the translocation pore.It has been shown for E. coli that cargos can be rejected from the SRP pathway during all targeting steps. In the first project, our interest concentrates on ribosomes translating the EspP signal sequence (RNCEspP). In vivo, EspP is a post-translationally targeted protein, but RNCEspP has been shown to be bound by SRP and FtsY leading to a non-productive “early”-like RNCEspP-SRP-FtsY complex. Using single particle cryo-electron microscopy (EM), we analysed the structural basis for the rejection of RNCEspP by SRP and FtsY. Comparison of our RNCEspP-SRP-FtsY cryo-EM structure to other available cryo-EM structures of co-translational targeting complexes containing the correct cargo RNCFtsQ unravelled differences in the SRP-FtsY structure between a correct cargo and an incorrect cargo. Two major differences between the targeting complexes containing the cargos RNCFtsQ and RNCEspP were observed: first, the Ffh M-domain was attached to ribosomal RNA helix 59 of RNCEspP, while it was detached from this site in the case of RNCFtsQ. It could be that such an ordered M-domain is hampering the release of the signal sequence which is required for successful completion of targeting. Second, the Ffh-FtsY NG-domain arrangement was flexible in the complex with RNCEspP in comparison to RNCFtsQ indicating that the "early"-like complex formed on RNCEspP is less stable. Biochemical data using fluorescence resonance energy transfer corroborated these results, showing that FtsY is bound with lower affinity in the RNCEspP “early” complex and that the rearrangement to the “closed” conformation is less efficient. Further biochemical analysis of EspP signal sequence variants showed that mainly the N-terminal extension of the EspP signal sequence is responsible for its rejection from the SRP pathway.

<p>Protein folding and export to the membrane are crucial events in the cell. Both processes may be initiated already at the ribosome, assisted by factors that bind to the polypeptide as it emerges from the ribosome. The signal recognition particle (SRP) scans the ribosome for nascent peptides destined for membrane insertion and targets these ribosomes to the site for translocation in the membrane. Trigger factor (TF) is a folding chaperone that interacts with nascent chains to promote their correct folding, prevent misfolding and aggregation. </p><p>In this thesis, we first investigated membrane targeting and insertion of two heterologous membrane proteins in E. coli by using in vitro translation, membrane targeting and cross-linking. We found that these proteins are dependent on SRP for targeting and that they initially interact with translocon components in the same way as native nascent membrane proteins. </p><p>Moreover we have characterised the SRP and TF interactions with the ribosome both with cross-linking experiments and with quantitative binding experiments. Both SRP and TF bind to ribosomal L23 close to the nascent peptide exit site where they are strategically placed for binding to the nascent polypeptide. </p><p>Quantitative analysis of TF and SRP binding determined their respective KD values for binding to non translating ribosomes and reveals that they bind simultaneously to the ribosome, thus having separate binding sites on L23. </p><p>Finally, binding studies on ribosome nascent chain adds clues as to how TF functions as a chaperone.</p>

Protein folding and export to the membrane are crucial events in the cell. Both processes may be initiated already at the ribosome, assisted by factors that bind to the polypeptide as it emerges from the ribosome. The signal recognition particle (SRP) scans the ribosome for nascent peptides destined for membrane insertion and targets these ribosomes to the site for translocation in the membrane. Trigger factor (TF) is a folding chaperone that interacts with nascent chains to promote their correct folding, prevent misfolding and aggregation. In this thesis, we first investigated membrane targeting and insertion of two heterologous membrane proteins in E. coli by using in vitro translation, membrane targeting and cross-linking. We found that these proteins are dependent on SRP for targeting and that they initially interact with translocon components in the same way as native nascent membrane proteins. Moreover we have characterised the SRP and TF interactions with the ribosome both with cross-linking experiments and with quantitative binding experiments. Both SRP and TF bind to ribosomal L23 close to the nascent peptide exit site where they are strategically placed for binding to the nascent polypeptide. Quantitative analysis of TF and SRP binding determined their respective KD values for binding to non translating ribosomes and reveals that they bind simultaneously to the ribosome, thus having separate binding sites on L23. Finally, binding studies on ribosome nascent chain adds clues as to how TF functions as a chaperone.

Translocation across or integration into the rough endoplasmic reticulum (RER) membrane is the first step in the intracelluar sorting of proteins in eukaryotic cells. This process is initiated when a signal sequence in the nascent protein chain emerges from the ribosome and is recognized by the signal recognition particle (SRP). The resulting SRP-ribosome-nascent chain-complex (SRP-RNC) is targeted to the RER membrane through the concerted action of the SRP and the SRP receptor (SR). The nascent chain is then displaced from SRP and transferred to the translocon, a proteinaceous channel composed of oligomers of the Sec61 complex. To gain a better understanding of the molecular mechanism of protein translocation, we treated ribosome-stripped micro somes with proteases of different cleavage specificities to sever cytoplasmic domains of SRα, SRβ, TRAM, and the Sec61 complex, and then characterized protein translocation intermediates that accumulate when Sec61α or SRβ is inactivated by proteolysis. We found that GTP hydrolysis by the SRα-SRP complex and dissociation of SRP54 from the signal sequence are blocked in the absence of a functional Sec61 complex. Experiments using SR-reconstituted proteoliposomes confirmed the assembly of a membrane-bound, GTP-stabilized post-targeting intermediate. These results strongly suggest that the Sec61 complex regulates the GTP hydrolysis cycle of the SRP-SR complex at the stage of signal sequence dissociation from SRP54. This regulatory role of Sec61α is proposed to provide a mechanism that inhibits signal sequence dissociation from SRP54 if the adjacent Sec61 complex is occupied by a translating ribosome, thereby insuring efficient transfer of an RNC from the SRP-SR complex to the translocation channel. We also found that complex formation between SRα and SRP is compromised in the absence of intact SRβ. Results obtained using a soluble system of in vitro translated SRα and SRβ suggest that SRβ is either required for GTP binding to SRα and SRP54 or for stabilizing the SRα-SRP complex. Moreover, using the XTP mutants of SRα and SRP54, we found that XTP cannot support efficient protein translocation in the absence of GTP. The addition of GTP dramatically promotes protein translocation into the endoplasmic reticulum, suggesting the GTPase activity of SRβ is required for this process. Further mutagenesis experiments revealed that the GTP-binding pocket of SRβ is involved in dimerization with SRa. All these data demonstrate that SRβ is important in protein translocation and will help elucidate the precise role of SRβ in vivo.

Yes / The ribosome exit site is a focal point for the interaction of protein-biogenesis factors that guide the fate of nascent polypeptides. These factors include chaperones such as NAC, N-terminal-modifying enzymes like Methionine aminopeptidase (MetAP), and the signal recognition particle (SRP), which targets secretory and membrane proteins to the ER. These factors potentially compete with one another in the short time-window when the nascent chain first emerges at the exit site, suggesting a need for regulation. Here, we show that MetAP contacts the ribosome at the universal adaptor site where it is adjacent to the α subunit of NAC. SRP is also known to contact the ribosome at this site. In the absence of NAC, MetAP and SRP antagonize each other, indicating a novel role for NAC in regulating the access of MetAP and SRP to the ribosome. NAC also functions in SRP-dependent targeting and helps to protect substrates from aggregation before translocation. / This work was supported by grants from the BBSRC [H007202/1] and Wellcome Trust [097820/Z/11/A].

Cotranslational translocation is initiated by targeting of a ribosome-bound nascent polypeptide chain (RNC) to the endoplasmic reticulum (ER) membrane. The targeting reaction is coordinated by the signal recognition particle (SRP) through its interaction with the RNC and the membrane-bound SRP receptor (SR). A vacant translocon is a prerequisite for the subsequent nascent chain release from SRP-SR-RNC complex. It has been proposed that the protease-accessible cytosolic domains of the Sec61p complex play an important role in posttargeting steps by providing the binding site for the ribosome or interacting with the SR to initiate the signal sequence releasing. In this study, we have investigated the detailed mechanism that allows transfer of the ribosome-nascent chain (RNC) from the SRP-SR complex to the translocon using yeast S. cerevisiaeas the model system. Point mutations in cytoplasmic loops six (L6) and eight (L8) of yeast Sec61p cause reductions in growth rates and defects in translocation of nascent polypeptides that utilize the cotranslational translocation pathway. Sec61 heterotrimers isolated from the L8 sec61 mutants have a greatly reduced affinity for 80S ribosomes. Cytoplasmic accumulation of protein precursors demonstrates that the initial contact between the large ribosomal subunit and the Sec61 complex is important for efficient insertion of a nascent polypeptide into the translocation pore. In contrast, point mutations in L6 of Sec61p inhibit cotranslational translocation without significantly reducing the ribosome binding activity, indicating that the L6 and L8 sec61mutants impact different steps in the cotranslational translocation pathway. An interaction between the signal recognition particle receptor (SR) and the Sec61 complex has been proposed to facilitate transfer of the ribosome-nascent chain (RNC) complex to an unoccupied translocon. The slow growth and cotranslational translocation defects caused by deletion of the transmembrane span of yeast SRβ (srp102pΔTMD) are exaggerated upon disruption of the SSH1 gene, which encodes the pore subunit of a cotranslational translocation channel. Disruption of the SBH2 gene, which encodes the β-subunit of the Ssh1p complex, likewise causes a synthetic growth defect when combined with srp102pΔTMD. The in vivo kinetics of translocon gating by RNCs were slow and inefficient in the ssh1Δ srp102pΔTMD mutant. A critical role for translocon β-subunits in SR recognition is supported by the observation that deletion of both translocon β-subunits causes a block in the cotranslational targeting pathway that resembles elimination of either subunit of the SR, and could be partially suppressed by expression of carboxy-terminal Sbh2p fragments.