Voltage sensitivity of the bacterial protein translocation channel
Sprache des Vortragstitels:
The heterotrimeric protein translocation channel SecYEG enables (i) soluble proteins to cross the inner membrane and (ii) hydrophobic proteins to enter the membrane interior. It contains an aqueous pore that in its resting state is sealed by a ring of six hydrophobic residues and a half helix (the plug) (Saparov et al., 2007). Signal sequence binding or ribosome binding both dislocate the plug and break the ring, thereby opening the channel to ions (Knyazev et al., 2013). The membrane barrier to ions is preserved because physiological values of the transmembrane potential close the channel by a yet unknown mechanism (Knyazev et al., 2014). Here we demonstrate that this voltage sensitivity does not depend on the ligand. That is, the open time decreases similarly with voltage for SecYEG channels that are bound to (i) signal peptides, (ii) translocation intermediates (proOmpA) and the motor protein SecA, (iii) a ribosome nascent-chain (FtsQ) complex or (iv) empty ribosomes. The observations were made with planar lipid bilayers that contained the purified and reconstituted SecYEG complex. They indicate that the voltage sensor must be part of the SecYEG channel. In search for the sensor we mutated charged residues, deleted the plug and performed various cross-link experiments, the outcome of which will be discussed.
Saparov, S.M., Erlandson, K., Cannon, K., Schaletzky, J., Schulman, S., Rapoport, T.A., and Pohl, P. (2007). Determining the Conductance of the SecY Protein Translocation Channel for Small Molecules. Mol. Cell 26, 501-509.
Knyazev, D.G., Lents, A., Krause, E., Ollinger, N., Siligan, C., Papinski, D., Winter, L., Horner, A., and Pohl, P. (2013). The Bacterial Translocon SecYEG Opens upon Ribosome Binding. J. Biol. Chem. 288, 17941-17946.
Knyazev, D.G., Winter, L., Bauer, B.W., Siligan, C., and Pohl, P. (2014). Ion Conductivity of the Bacterial Translocation Channel SecYEG Engaged in Translocation. J. Biol. Chem. 289, 24611-24616.