Proton gradients in bacterial protein translocation
Sprache der Bezeichnung:
The bacterial translocon SecYEG is a key protein responsible for translocation across or reconstitution into the cytoplasmic membrane of most secretory and membrane proteins. The energy for peptide chain translocation may stem from ATP or GTP hydrolysis. That is, SecA or ribosome may push the polypeptide chain through the SecYEG in post-translational or co-translational mode of translocation, respectively. Alternatively or in addition to that, the transmembrane electrochemical proton gradient (proton motive force) may drive translocation. The corresponding molecular mechanisms are unknown. Here we focus on the molecular mechanism of proton-driven translocation. To decipher this, we will reconstitute co- and post-translational translocation intermediates into the free-spanning planar bilayer thereby gaining access to both periplasmic and cytoplasmic sides of the translocon. This, together with a combination of single molecule dye tracing and electrophysiological techniques, will for the first time allow straightforward control over transmembrane potential and pH gradient. We aim to distinguish between the two basic hypotheses, where: (1) the pH gradient controls the protonation state of the titratable residues in the peptide chain or in the translocon itself thereby controlling the chain?s movement through the translocon and (2) the pH gradient locally changes the transmembrane potential thereby affecting translocation. Clarifying this aspect of the translocation would allow finding strategies for efficiently manipulating recombinant protein production rate for biotechnological and research purposes (e.g. for studying membrane protein folding).