Previously, the role of YidC in the membrane protein biogenesis of the F 0 sector of the Escherichia coli F 1 F 0 ATP synthase was investigated. Whereas subunits a and c of the F 1 F 0 ATP synthase were strictly dependent on YidC for membrane insertion, subunit b required YidC for efficient insertion (Yi, L., Jiang, F., Chen, M., Cain, B., Bolhuis, A., and Dalbey, R. E. (2003) Biochemistry 42, 10537-10544). In this paper, we investigated other protein components and energetics that are required in the membrane protein assembly of the F 0 sector subunits. We show here that the Sec translocase and the signal recognition particle (SRP) pathway are required for membrane insertion of subunits a and b. In contrast, subunit c required neither the Sec machinery nor the SRP pathway for insertion. While the proton motive force was not required for insertion of subunits b and c, it was required for translocation of the negatively charged periplasmic NH 2 -terminal tail of subunit a, whereas periplasmic loop 2 of subunit a could insert in a proton motive force-independent manner. Taken together, the in vivo data suggest that subunits a and b are inserted by the Sec/SRP pathway with the help of YidC, and subunit c is integrated into the membrane by the novel YidC pathway.The insertion of bacterial inner membrane proteins can occur by two pathways, one involving the Sec translocase and the other independent of the Sec translocase. The Sec translocase is comprised of the protein-conducting SecYEG channel (1) and the heterotrimeric SecDFYajC complex (2) that facilitates translocation. Interestingly, the Sec complex is also associated with YidC (3), a key component that plays an important role in membrane protein biogenesis (4, 5). YidC mediates membrane insertion and comes into contact with the hydrophobic regions of Sec-dependent membrane proteins (6, 7). For translocation of large domains of membrane proteins, SecA is also needed (8 -10). Typically, membrane proteins are targeted to the Sec translocase by the SRP 1 pathway (11). Sec-independent membrane proteins use the YidC pathway for insertion. The M13 procoat and Pf3 coat proteins, which insert by a Sec-independent mechanism (12, 13), strictly require YidC for their membrane insertion (14 -16). In addition, photocross-linking studies have shown that YidC plays a direct role as YidC is cross-linked to the Pf3 coat during membrane insertion (16). No contacts were observed between the Pf3 coat and the Sec components. While insertion of the Pf3 coat and M13 procoat proteins is dependent on the proton motive force (pmf) (13, 17), they do not require the SRP components for targeting (16,18).Recently, it has been shown that YidC is involved in the membrane assembly of cytochrome bo3 oxidase and the F 1 F 0 ATP synthase but not NADH-dehydrogenase (19). These multisubunit membrane complexes play vital roles in cellular respiration where they participate in the complex chemistry of electron transport and coupled oxidative phosphorylation. When YidC is depleted, there is a measurabl...

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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.

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