Interaction of the SecYEG translocon with the SRP receptor and the ribosome
by Albena Draycheva
Date of Examination:2014-05-16
Date of issue:2015-05-08
Advisor:Prof. Dr. Wolfgang Wintermeyer
Referee:Prof. Dr. Wolfgang Wintermeyer
Referee:Prof. Dr. Kai Tittmann
Referee:Prof. Dr. Marina Bennati
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Abstract
English
This work aimed at studying the interaction between SecYEG and FtsY and before and after the translating ribosome has been transferred to the SecYEG translocon. In this study we have used monomers of the SecYEG complex embedded into nanodiscs (SecYEG(ND)) in combination with fluorescence measurements, in particular fluorescence resonance energy transfer (FRET), to determine the affinity of FtsY binding to SecYEG (Kd 0.18 ± 0.02 µM). Using a rapid kinetics approach we verified that FtsY is primarily localized at the membrane and that it interacts with SecYEG(ND) via two interaction sites. The binding of FtsY to SecYEG(ND) is mediated via both the NG and the A domains. The A domain, especially the first 208 amino acids assure the stable binding of FtsY. Further, we have demonstrated that the A and NG domain of FtsY are strongly bound to one another keeping FtsY in a ‘closed’ conformation when bound to the membrane. The interaction of FtsY with SecYEG(ND) induces a rearrangement between the domains and FtsY adopts an ‘open’ conformation, which would facilitate the efficient binding of the FtsY-NG domain to the homologous domain of SRP. Due to the high affinity of the complex of translating ribosomes and SecYEG(ND), and the increased affinity of ribosome-bound SRP to FtsY the ribosome-SRP complex is localized properly at the site of the translocon. After the ribosome has been transferred to SecYEG and SRP and FtsY have been separated due to GTP hydrolysis, FtsY remains associated with the SecYEG complex to take part in another round of targeting. Our findings also show that the A domain of FtsY mainly takes part in the stabilization of FtsY on the translocon before and after ribosomal transfer, but is not essential for GTPase activation. By contrast, the FtsY-NG domain is responsible for GTP-dependent complex formation with SRP.
Keywords: translocon; signal recognition particle; signal recognition particle receptor; ribosome; cotranslational; membrane proteins; nanodiscs; fluorescence