The Sec translocase: structure function analysis in protein translocation and membrane protein insertion

PhD ceremony: Mr. D.J.F. du Plessis, 13.15 uur, Academiegebouw, Broerstraat 5, Groningen

Title: The Sec translocase: structure function analysis in protein translocation and membrane protein insertion

Promotor(s): prof. A.J.M. Driessen

Faculty: Mathematics and Natural Sciences

In the thesis of François du Plessis a structure-function analysis has been made of the SecYEG translocase with regards to its role in translocation and insertion of various proteins. By utilizing in vitro protein translocation assays it was found that the translocon and YidC act cooperatively to insert an essential subunit of the cytochrome oxidase (CyoA) into the inner membrane. Following the recent availability of a crystal structure of the Sec translocase, directed cysteine crosslinking with crosslinkers of various lengths has been utilized to investigate the role of the proposed lateral gate of the translocase. Here it was shown that opening of the lateral gate is not only essential during protein translocation but directly linked to the activity of the motor protein, SecA, suggesting an allosteric mechanism for the opening of the lateral gate. Interestingly, it was found that the insertion of transmembrane domains do not require the opening of the lateral gate, suggesting a unique release mechanism for transmembrane domains. The functional role for higher order structures of the translocon was also further investigated. Here, directed cysteine crosslinking has been employed to crosslink two translocons in a front-to-front manner while excluding the formation of a consolidated pore. Interestingly, it was found that the front-to-front crosslinking of translocons did not impair protein translocation or membrane protein insertion. The findings of this thesis are discussed in context with regards to current knowledge on the translocase structure and function.