Dimeric E. coli YidC forms an ion-condunting pore in the membrane
Sprache des Vortragstitels:
Mechanisms and Regulation of Protein Translocation
Sprache des Tagungstitel:
The universally conserved protein YidC functions as a membrane protein insertase. It (i) facilitates the exit of transmembrane domains at the lateral gate of SecYEG and (ii) inserts short or closely spaced membrane proteins independently of SecYEG. According to the recent X-ray structure of B. halodurans YidC in monoolein, a monomeric YidC harbors a hydrophilic groove in the middle of the membrane (Kumazaki et al., Nature, 2014). A cryo-EM density map of RNC bound YidC in detergent also suggests that YidC functions as a monomer (Wickles et al., eLife, 2014). However, it is unclear how the membrane barrier to protons and other ions may be maintained with such a hydrophilic groove at the protein lipid interface in the middle of the membrane. Here we have tested whether YidC may maintain this barrier on its own or whether accessory molecules are required. We overexpressed YidC and reconstituted the purified protein into vesicular lipid bilayers formed from E. coli polar lipid extract. Fluorescence correlation spectroscopy experiments with labeled and reconstituted YidC indicate the formation of dimers in this lipid composition. This assumption is also supported by BN-PAGE of YidC in inverted membrane vesicles, which show a dimeric YidC in the absence of SDS.
When reconstituted into free-standing planar lipid bilayers YidC conserved the membrane barrier to protons and ions under an applied transmembrane voltage. Furthermore, electrophysiological experiments revealed that YidC is able to form an ion conducting pore in the membrane, which is opened by ribosome binding alike that of SecYEG (Knyazev et al., JBC, 2013). Channel conductivity suggests that YidC has a smaller water-filled lumen than the SecYEG channel (Saparov et al., Mol.Cell., 2007). The lumen would still be wide enough to accommodate an ?-helix.