The F-0 c subunit of F0F1 ATPase (F-0-c) possesses two membrane-spanning stretches with N- and C-termini exposed to the periplasmic (extracellular) side of the cytoplasmic membrane of E. coli. Although F-0-c insertion has been extensively analyzed in vitro by means of protease protection assaying, it is unclear whether such assays allow elucidation of the insertion process faithfully, since the membrane protected fragment, an index of membrane insertion, is a full-length polypeptide of F-0-c, which is the same as the protease-resistant conformation without membrane insertion. We found that the protease-resistant conformation could be discriminated from membrane-insertion by including octyl glucoside on protease digestion. By means of this system, we found that F-0-c insertion depends on MPlase, a glycolipozyme involved in membrane insertion, and is stimulated by YidC. In addition, we found that acidic phospholipids PG and CL transform F-0-c into a protease-resistant form, while MPIase prevents the acquisition of such a protease-resistant conformation. (C) 2017 Elsevier Inc. All rights reserved.