Mechanism and Hydrophobic Forces Driving Membrane Protein Insertion of Subunit II of Cytochrome Bo 3 Oxidase

Subunit II (CyoA) of cytochrome bo(3) oxidase, which spans the inner membrane twice in bacteria, has several unusual features in membrane biogenesis. It is synthesized with an amino-terminal cleavable signal peptide. In addition, distinct pathways are used to insert the two ends of the protein. The amino-terminal domain is inserted by the YidC pathway whereas the large carboxyl-terminal domain is translocated by the SecYEG pathway. Insertion of the protein is also proton motive force (pmf)-independent. Here we examined the topogenic sequence requirements and mechanism of insertion of CyoA in bacteria. We find that both the signal peptide and the first membrane-spanning region are required for insertion of the amino-terminal periplasmic loop. The pmf-independence of insertion of the first periplasmic loop is due to the loop's neutral net charge. We observe also that the introduction of negatively charged residues into the periplasmic loop makes insertion pmf dependent, whereas the addition of positively charged residues prevents insertion unless the pmf is abolished. Insertion of the carboxyl-terminal domain in the full-length CyoA occurs by a sequential mechanism even when the CyoA amino and carboxyl-terminal domains are swapped with other domains. However, when a long spacer peptide is added to increase the distance between the amino-terminal and carboxyl-terminal domains, insertion no longer occurs by a sequential mechanism.