Both YidC and SecYEG are required for translocation of the periplasmic loops 1 and 2 of the multispanning membrane protein TatC.

TatC, a subunit of the twin arginine translocase, is a 6-membrane-spanning protein exposing three periplasmic loops. We have used TatC as a model system to examine how multispanning proteins insert into the membrane. To assay translocation of each of the three loops of TatC across the membrane, we used trypsin mapping, proteinase K mapping, and chemical modification methods. Here, we show that the signal recognition particle is required for targeting TatC to the inner membrane of Escherichiacoli. While translocation of loops 1 and 2 is strictly dependent on the Sec translocase and the YidC insertase, translocation of loop 3 does not depend on the translocase or insertase. None of the periplasmic loops require SecA or the proton motive force for membrane translocation. This work demonstrates a strategy where all the loops of a multispanning membrane protein can be monitored individually. The membrane translocation mechanism of each periplasmic loop can be complex with different energy and translocase requirements for a multispanning membrane protein.