In vitro studies on the SecA involvement in the assembly of an integral membrane protein with large hydrophilic domain

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.

Author dc:creator

• Antonoaea, Raluca

Contributors dc:contributor

• Müller, Matthias

• SecYEG Translokon, E. coli, Ribosomen-assoziierte naszierende Ketten, Translokationsintermediate
• SecA, ribosome-associated nascent chains, E.coli, translocation intermediates, PpiD

Repository record source_url

https://freidok.uni-freiburg.de/data/2371

OAI identifier oai:identifier

oai:freidok.uni-freiburg.de:2371