YidC from Escherichia coli Forms an Ion-Conducting Pore upon Activation by Ribosomes

Area of Science:

• Membrane biology
• Protein biophysics
• Microbiology

Background:

• The universally conserved YidC protein is essential for inserting and folding transmembrane polypeptides.
• Current models suggest a charged arginine residue facilitates polypeptide sliding along YidC's surface.
• The mechanism by which YidC maintains membrane integrity during this process remains unclear.

Purpose of the Study:

• To investigate the structural and functional properties of E. coli YidC.
• To determine if YidC forms a pore and how this relates to its function in protein insertion.
• To explore the oligomeric state of YidC and its implications for pore formation.

Main Methods:

• Purification and reconstitution of E. coli YidC.
• AlphaFold modeling to predict YidC dimer structure.
• Blue-native polyacrylamide gel electrophoresis (BN-PAGE) of native vesicles.
• Fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence photobleaching.
• Crosslinking experiments to confirm YidC assembly.

Main Results:

• Purified and reconstituted YidC forms an ion-conducting transmembrane pore upon binding to ribosomes or ribosome-nascent chain complexes (RNCs).
• An AlphaFold model of a YidC dimer revealed a pore structure, contrasting with monomeric YidC.
• Experimental data from BN-PAGE, FCS, photobleaching, and crosslinking supported a dimeric assembly of YidC.
• In the dimeric model, key residues like arginine point into the pore, interacting with nascent chains.

Conclusions:

• E. coli YidC forms a dimeric, ion-conducting pore structure upon ribosome binding.
• This pore formation provides a potential mechanism for maintaining membrane integrity during protein insertion.
• The findings suggest an alternative YidC-assisted insertion mode distinct from the traditional insertase mechanism.