{"id":{"repo_id":"freiburg-diss","oai_identifier":"oai:freidok.uni-freiburg.de:2371"},"canonical_url":"https://search.dev.ndltd.org/etd/freiburg-diss/oai:freidok.uni-freiburg.de:2371","repository":{"repo_id":"freiburg-diss","name":"University of Freiburg","base_url":"https://freidok.uni-freiburg.de/oai/oai2.php"},"display":{"title":"In vitro studies on the SecA involvement in the assembly of an integral membrane protein with large hydrophilic domain","abstract":"The transport of secretory proteins across the inner membrane of E. coli as well as the integration of inner membrane proteins takes place via the SecYEG-translocon. The targeting pathways of secretory and membrane proteins to the SecYEG translocon however, are different. Integral membrane proteins are co-translationally targeted by signal recognition particle (SRP) and its receptor FtsY, while secretory proteins follow a post-translational targeting mode mediated by SecB/SecA. The subsequent transport, of secretory proteins across the membrane depends on the ATPase activity of SecA. Integral membrane proteins with large hydrophilic domains require the concomitant actions of both SRP/FtsY for targeting and SecA for the transport of the hydrophilic domains across the membrane. The work presented in this thesis focuses on the understanding of how SecA functions at a post targeting level in the assembly of inner membrane proteins with large hydrophilic domains. <br> We used as a substrate ribosome-associated nascent chains of Momp2. Momp2 is a hybrid protein encompassing an N-terminal transmembrane domain and a large translocated moiety. Momp2 is one of the proteins whose integration via the transmembrane domain takes place in a SRP/FtsY dependent manner, whereas the transport of its hydrophilic domain requires the help of SecA. Previous studies demonstrated that the transport of differently sized nascent Momp2 polypeptides was promoted by SecA, provided that the ribosome was released from the polypeptide by puromycin. <br>We could show in this thesis that SecA promotes a partial translocation of nascent Momp2 chains even if they are still attached to the ribosome. This partial translocation became manifest after treatment of the membrane-bound nascent Momp2 chains with proteinase K yielding two prominent protease-protected fragments of 16 and 26 kDa. Because these fragments were chased to a full-size translocation product of Momp2 after release of the ribosome by puromycin they must represent translocation intermediates. The 16 and 26 kDa fragments were formed in the presence of active SecA. Furthermore, it is shown that the proton motive force and SecG play an important role in the SecA-dependent accumulation of those translocation intermediates of nascent Momp2 chains. <br> For small ribosome-associated nascent Momp2 chains the translocation intermediates similar translocation intermediates could not be detected after proteinase K treatment. However, site directed cross-linking revealed contacts between the hydrophilic domain of short nascent Momp2 chains and SecY indicating that even small ribosome-associated chains are partially translocated in a SecA-dependent manner. <br>Finally, it is demonstrated that SecA pushes nascent Momp2 chains across the SecYEG translocon directly to the periplasmic chaperone PpiD, while the most C-terminal parts are still trapped in the translocon due to their attachment to the ribosome.","abstract_html":"The transport of secretory proteins across the inner membrane of E. coli as well as the integration of inner membrane proteins takes place via the SecYEG-translocon. The targeting pathways of secretory and membrane proteins to the SecYEG translocon however, are different. Integral membrane proteins are co-translationally targeted by signal recognition particle (SRP) and its receptor FtsY, while secretory proteins follow a post-translational targeting mode mediated by SecB/SecA. The subsequent transport, of secretory proteins across the membrane depends on the ATPase activity of SecA. Integral membrane proteins with large hydrophilic domains require the concomitant actions of both SRP/FtsY for targeting and SecA for the transport of the hydrophilic domains across the membrane. The work presented in this thesis focuses on the understanding of how SecA functions at a post targeting level in the assembly of inner membrane proteins with large hydrophilic domains. &lt;br&gt; We used as a substrate ribosome-associated nascent chains of Momp2. Momp2 is a hybrid protein encompassing an N-terminal transmembrane domain and a large translocated moiety. Momp2 is one of the proteins whose integration via the transmembrane domain takes place in a SRP/FtsY dependent manner, whereas the transport of its hydrophilic domain requires the help of SecA. Previous studies demonstrated that the transport of differently sized nascent Momp2 polypeptides was promoted by SecA, provided that the ribosome was released from the polypeptide by puromycin. &lt;br&gt;We could show in this thesis that SecA promotes a partial translocation of nascent Momp2 chains even if they are still attached to the ribosome. This partial translocation became manifest after treatment of the membrane-bound nascent Momp2 chains with proteinase K yielding two prominent protease-protected fragments of 16 and 26 kDa. Because these fragments were chased to a full-size translocation product of Momp2 after release of the ribosome by puromycin they must represent translocation intermediates. The 16 and 26 kDa fragments were formed in the presence of active SecA. Furthermore, it is shown that the proton motive force and SecG play an important role in the SecA-dependent accumulation of those translocation intermediates of nascent Momp2 chains. &lt;br&gt; For small ribosome-associated nascent Momp2 chains the translocation intermediates similar translocation intermediates could not be detected after proteinase K treatment. However, site directed cross-linking revealed contacts between the hydrophilic domain of short nascent Momp2 chains and SecY indicating that even small ribosome-associated chains are partially translocated in a SecA-dependent manner. &lt;br&gt;Finally, it is demonstrated that SecA pushes nascent Momp2 chains across the SecYEG translocon directly to the periplasmic chaperone PpiD, while the most C-terminal parts are still trapped in the translocon due to their attachment to the ribosome.","abstract_has_math":false,"creators":["Antonoaea, Raluca"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["Müller, Matthias"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":null,"date_issued":"","date_published":null,"updated_at":"2026-07-24T02:22:46Z","subjects":["SecYEG Translokon, E. coli, Ribosomen-assoziierte naszierende Ketten, Translokationsintermediate","SecA, ribosome-associated nascent chains, E.coli, translocation intermediates, PpiD"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://freidok.uni-freiburg.de/data/2371","outbound_label":"Repository record","outbound_source":"source_url"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Müller, Matthias"]},{"key":"dc:creator","label":"Author","values":["Antonoaea, Raluca"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:type","label":"Dc Type","values":["DoctoralThesis"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["SecYEG Translokon, E. coli, Ribosomen-assoziierte naszierende Ketten, Translokationsintermediate","SecA, ribosome-associated nascent chains, E.coli, translocation intermediates, PpiD"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["The transport of secretory proteins across the inner membrane of E. coli as well as the integration of inner membrane proteins takes place via the SecYEG-translocon. The targeting pathways of secretory and membrane proteins to the SecYEG translocon however, are different. Integral membrane proteins are co-translationally targeted by signal recognition particle (SRP) and its receptor FtsY, while secretory proteins follow a post-translational targeting mode mediated by SecB/SecA. The subsequent transport, of secretory proteins across the membrane depends on the ATPase activity of SecA. Integral membrane proteins with large hydrophilic domains require the concomitant actions of both SRP/FtsY for targeting and SecA for the transport of the hydrophilic domains across the membrane. The work presented in this thesis focuses on the understanding of how SecA functions at a post targeting level in the assembly of inner membrane proteins with large hydrophilic domains. <br> We used as a substrate ribosome-associated nascent chains of Momp2. Momp2 is a hybrid protein encompassing an N-terminal transmembrane domain and a large translocated moiety. Momp2 is one of the proteins whose integration via the transmembrane domain takes place in a SRP/FtsY dependent manner, whereas the transport of its hydrophilic domain requires the help of SecA. Previous studies demonstrated that the transport of differently sized nascent Momp2 polypeptides was promoted by SecA, provided that the ribosome was released from the polypeptide by puromycin. <br>We could show in this thesis that SecA promotes a partial translocation of nascent Momp2 chains even if they are still attached to the ribosome. This partial translocation became manifest after treatment of the membrane-bound nascent Momp2 chains with proteinase K yielding two prominent protease-protected fragments of 16 and 26 kDa. Because these fragments were chased to a full-size translocation product of Momp2 after release of the ribosome by puromycin they must represent translocation intermediates. The 16 and 26 kDa fragments were formed in the presence of active SecA. Furthermore, it is shown that the proton motive force and SecG play an important role in the SecA-dependent accumulation of those translocation intermediates of nascent Momp2 chains. <br> For small ribosome-associated nascent Momp2 chains the translocation intermediates similar translocation intermediates could not be detected after proteinase K treatment. However, site directed cross-linking revealed contacts between the hydrophilic domain of short nascent Momp2 chains and SecY indicating that even small ribosome-associated chains are partially translocated in a SecA-dependent manner. <br>Finally, it is demonstrated that SecA pushes nascent Momp2 chains across the SecYEG translocon directly to the periplasmic chaperone PpiD, while the most C-terminal parts are still trapped in the translocon due to their attachment to the ribosome.","Der Transport von sekretorischen Proteinen über die innere <br>Membran von E. coli sowie die Integration von <br>Membranproteinen erfolgt über das SecYEG Translokon. Die <br>Art und Weise der Zielsteuerung von sekretorischen und <br>Membranproteinen an das SecYEG Translokon weist jedoch <br>erhebliche Unterschiede auf. So werden Membranproteine <br>kotranslational durch den Signalerkennungspartikel (SRP) <br>und dessen Rezeptor FtsY an das Sec Translokon <br>transportiert, während sekretorische Proteine einem <br>posttranslationalem Zielsteuerungsmodus folgen, der durch <br>SecB/SecA vermittelt wird. Der darauf folgende Transport <br>von sekretorischen Proteinen über die Membran hinweg ist <br>abhängig von der ATPase-Aktivität des SecA. <br>Membranproteine, die eine große hydrophile Domäne <br>beinhalten, erfordern gleichzeitig sowohl SRP/FtsY für die <br>Zielsteuerung als auch SecA für den Transport der <br>hydrophilen Domäne über die Membran. Ziel dieser Arbeit war <br>die Aufklärung der SecA Funktion während einer <br>posttranslationalen Zielsteuerung bei der Assemblierung von <br>Membranproteinen, die große hydrophile Domänen aufweisen. <br>Als Substrat wurden Ribosomen-assoziierte naszierende <br>Ketten von Momp2 verwendet. Bei Momp2 handelt es sich um <br>ein Hybridprotein, das sowohl eine N-terminale <br>Transmembrandomäne als auch einen großen zu <br>translozierenden Teil umfasst. Momp2 stellt eines der <br>Proteine dar, bei denen die Integration der <br>Transmembrandomäne in einer SRP/FtsY-abhängigen Weise <br>vollzogen wird, wohingegen der Transport seiner hydrophilen <br>Domäne durch SecA vermittelt wird. Bisherige Studien <br>zeigten, dass der Transport von verschieden großen <br>naszierenden Momp2 Polypeptiden durch SecA gefördert wurde, <br>unter der Voraussetzung, dass das Ribosom durch Puromycin <br>von der Polypetidkette abgelöst wurde. <br>In dieser Arbeit konnte gezeigt werden, dass SecA die <br>Translokation von naszierenden Momp2 Ketten teilweise <br>begünstigt, selbst wenn diese noch mit dem Ribosom <br>assoziiert sind. Diese partielle Translokation wurde nach <br>der Behandlung von Membran-gebundenen naszierenden Momp2 <br>Ketten mit Proteinase K sichtbar; als Resultat zeigten sich <br>zwei Protease-geschützte Fragmente mit einer Größe von 16 <br>und 26 kDa. Da man die Bildung eines vollständigen <br>Translokationsproduktes von Momp2 nach der Entlassung des <br>Ribosoms durch Puromycin verfolgen konnte, müssen diese <br>Fragmente Translokationsintermediate repräsentieren. Die <br>beiden 16 und 26 kDa Fragmente wurden in Anwesenheit von <br>aktiven SecA gebildet. Weiterhin wurde gezeigt, dass die <br>protonenmotorische Kraft und SecG eine wichtige Rolle in <br>der SecA-abhängigen Anhäufung dieser <br>Translokationsintermediate spielen. <br>Für kleinere Ribosomen-assoziierte naszierende Momp2 Ketten <br>konnten keine solche Translokationsintermediate nach <br>Proteinase K-Behandlung gefunden werden. Dennoch zeigten <br>spezifische Crosslinking Studien, dass zwischen der <br>hydrophilen Domäne von kurzen naszierenden Momp2 Ketten und <br>SecY Kontakte ausgebildet werden konnten. Dies weist darauf <br>hin, dass sogar kleine Ribosomen-assoziierte Ketten in <br>einer SecA-abhängigen Weise teilweise transloziert werden <br>können. <br>Schließlich konnte auch dargelegt werden, dass SecA die <br>naszierenden Momp2 Ketten über das SecYEG Translokon direkt <br>zu dem periplasmatischen Chaperon PpiD führt, während die <br>äußersten C-terminalen Teile immer noch im Translokon <br>arretiert sind aufgrund ihrer Assoziation mit dem Ribosom."]},{"key":"dc:format.medium","label":"Dc Format Medium","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["In vitro studies on the SecA involvement in the assembly of an integral membrane protein with large hydrophilic domain","In vitro Studien über die SecA-Beteiligung am Einbau eines integralen Membranproteins mit großer, hydrophiler Domäne"]}]}],"canonical_facts":{"dc:contributor":["Müller, Matthias"],"dc:creator":["Antonoaea, Raluca"],"dc:description.abstract":["The transport of secretory proteins across the inner membrane of E. coli as well as the integration of inner membrane proteins takes place via the SecYEG-translocon. The targeting pathways of secretory and membrane proteins to the SecYEG translocon however, are different. Integral membrane proteins are co-translationally targeted by signal recognition particle (SRP) and its receptor FtsY, while secretory proteins follow a post-translational targeting mode mediated by SecB/SecA. The subsequent transport, of secretory proteins across the membrane depends on the ATPase activity of SecA. Integral membrane proteins with large hydrophilic domains require the concomitant actions of both SRP/FtsY for targeting and SecA for the transport of the hydrophilic domains across the membrane. The work presented in this thesis focuses on the understanding of how SecA functions at a post targeting level in the assembly of inner membrane proteins with large hydrophilic domains. <br> We used as a substrate ribosome-associated nascent chains of Momp2. Momp2 is a hybrid protein encompassing an N-terminal transmembrane domain and a large translocated moiety. Momp2 is one of the proteins whose integration via the transmembrane domain takes place in a SRP/FtsY dependent manner, whereas the transport of its hydrophilic domain requires the help of SecA. Previous studies demonstrated that the transport of differently sized nascent Momp2 polypeptides was promoted by SecA, provided that the ribosome was released from the polypeptide by puromycin. <br>We could show in this thesis that SecA promotes a partial translocation of nascent Momp2 chains even if they are still attached to the ribosome. This partial translocation became manifest after treatment of the membrane-bound nascent Momp2 chains with proteinase K yielding two prominent protease-protected fragments of 16 and 26 kDa. Because these fragments were chased to a full-size translocation product of Momp2 after release of the ribosome by puromycin they must represent translocation intermediates. The 16 and 26 kDa fragments were formed in the presence of active SecA. Furthermore, it is shown that the proton motive force and SecG play an important role in the SecA-dependent accumulation of those translocation intermediates of nascent Momp2 chains. <br> For small ribosome-associated nascent Momp2 chains the translocation intermediates similar translocation intermediates could not be detected after proteinase K treatment. However, site directed cross-linking revealed contacts between the hydrophilic domain of short nascent Momp2 chains and SecY indicating that even small ribosome-associated chains are partially translocated in a SecA-dependent manner. <br>Finally, it is demonstrated that SecA pushes nascent Momp2 chains across the SecYEG translocon directly to the periplasmic chaperone PpiD, while the most C-terminal parts are still trapped in the translocon due to their attachment to the ribosome.","Der Transport von sekretorischen Proteinen über die innere <br>Membran von E. coli sowie die Integration von <br>Membranproteinen erfolgt über das SecYEG Translokon. Die <br>Art und Weise der Zielsteuerung von sekretorischen und <br>Membranproteinen an das SecYEG Translokon weist jedoch <br>erhebliche Unterschiede auf. So werden Membranproteine <br>kotranslational durch den Signalerkennungspartikel (SRP) <br>und dessen Rezeptor FtsY an das Sec Translokon <br>transportiert, während sekretorische Proteine einem <br>posttranslationalem Zielsteuerungsmodus folgen, der durch <br>SecB/SecA vermittelt wird. Der darauf folgende Transport <br>von sekretorischen Proteinen über die Membran hinweg ist <br>abhängig von der ATPase-Aktivität des SecA. <br>Membranproteine, die eine große hydrophile Domäne <br>beinhalten, erfordern gleichzeitig sowohl SRP/FtsY für die <br>Zielsteuerung als auch SecA für den Transport der <br>hydrophilen Domäne über die Membran. Ziel dieser Arbeit war <br>die Aufklärung der SecA Funktion während einer <br>posttranslationalen Zielsteuerung bei der Assemblierung von <br>Membranproteinen, die große hydrophile Domänen aufweisen. <br>Als Substrat wurden Ribosomen-assoziierte naszierende <br>Ketten von Momp2 verwendet. Bei Momp2 handelt es sich um <br>ein Hybridprotein, das sowohl eine N-terminale <br>Transmembrandomäne als auch einen großen zu <br>translozierenden Teil umfasst. Momp2 stellt eines der <br>Proteine dar, bei denen die Integration der <br>Transmembrandomäne in einer SRP/FtsY-abhängigen Weise <br>vollzogen wird, wohingegen der Transport seiner hydrophilen <br>Domäne durch SecA vermittelt wird. Bisherige Studien <br>zeigten, dass der Transport von verschieden großen <br>naszierenden Momp2 Polypeptiden durch SecA gefördert wurde, <br>unter der Voraussetzung, dass das Ribosom durch Puromycin <br>von der Polypetidkette abgelöst wurde. <br>In dieser Arbeit konnte gezeigt werden, dass SecA die <br>Translokation von naszierenden Momp2 Ketten teilweise <br>begünstigt, selbst wenn diese noch mit dem Ribosom <br>assoziiert sind. Diese partielle Translokation wurde nach <br>der Behandlung von Membran-gebundenen naszierenden Momp2 <br>Ketten mit Proteinase K sichtbar; als Resultat zeigten sich <br>zwei Protease-geschützte Fragmente mit einer Größe von 16 <br>und 26 kDa. Da man die Bildung eines vollständigen <br>Translokationsproduktes von Momp2 nach der Entlassung des <br>Ribosoms durch Puromycin verfolgen konnte, müssen diese <br>Fragmente Translokationsintermediate repräsentieren. Die <br>beiden 16 und 26 kDa Fragmente wurden in Anwesenheit von <br>aktiven SecA gebildet. Weiterhin wurde gezeigt, dass die <br>protonenmotorische Kraft und SecG eine wichtige Rolle in <br>der SecA-abhängigen Anhäufung dieser <br>Translokationsintermediate spielen. <br>Für kleinere Ribosomen-assoziierte naszierende Momp2 Ketten <br>konnten keine solche Translokationsintermediate nach <br>Proteinase K-Behandlung gefunden werden. Dennoch zeigten <br>spezifische Crosslinking Studien, dass zwischen der <br>hydrophilen Domäne von kurzen naszierenden Momp2 Ketten und <br>SecY Kontakte ausgebildet werden konnten. Dies weist darauf <br>hin, dass sogar kleine Ribosomen-assoziierte Ketten in <br>einer SecA-abhängigen Weise teilweise transloziert werden <br>können. <br>Schließlich konnte auch dargelegt werden, dass SecA die <br>naszierenden Momp2 Ketten über das SecYEG Translokon direkt <br>zu dem periplasmatischen Chaperon PpiD führt, während die <br>äußersten C-terminalen Teile immer noch im Translokon <br>arretiert sind aufgrund ihrer Assoziation mit dem Ribosom."],"dc:format.medium":["application/pdf"],"dc:subject":["SecYEG Translokon, E. coli, Ribosomen-assoziierte naszierende Ketten, Translokationsintermediate","SecA, ribosome-associated nascent chains, E.coli, translocation intermediates, PpiD"],"dc:title":["In vitro studies on the SecA involvement in the assembly of an integral membrane protein with large hydrophilic domain","In vitro Studien über die SecA-Beteiligung am Einbau eines integralen Membranproteins mit großer, hydrophiler Domäne"],"dc:type":["DoctoralThesis"]},"updated_at":"2026-07-24T02:22:46Z"}