Equilibrium Sampling between Membrane Interior and the Aqueous SecYEG Channel Departs from the Biological Hydrophobicity Scale
Sprache des Vortragstitels:
The bacterial translocon SecYEG either inserts polypeptides into the plasma membrane or translocates them into the periplasm. The decision is assumed to reflect true thermodynamic equilibrium. Here we tested the hypothesis by allowing a true translocation intermediate to sample the different environments of the hydrophobic membrane interior and the hydrophilic aqueous channel for long intervals of time. Therefore we reconstituted the purified SecYEG complex into planar lipid bilayers1 and monitored its interaction with the purified ribosome-nascent chain complex2. Calmodulin binding to a calmodulin binding tag at the N-terminus prevented backsliding of the translocation intermediate. Localization of the thus locked polypeptide into the aqueous channel resulted in ion channel activity, whereas its partitioning into the hydrophobic membrane interior led to channel closure. In case of hydrophilic polypeptides we observed only partial membrane partitioning - even at physiologically relevant voltages. The dwell time of hydrophobic chains in the SecY channel decreased with their hydrophobicity and with absolute values of membrane voltage. Yet it considerably exceeded the time that is allotted to a peptide segment for sampling the environment in vivo. Due to the short translocation time, moderately hydrophobic segments are likely to be secreted instead of being inserted into the membrane. Our observation partly explains, why SecYEG does not recognize outer membrane proteins (OMP). They require a second translocation machinery to be inserted into the membrane.