The YidC/Oxa1/Alb3 insertases are conserved in all domains of life,1, 2 playing crucial roles in the membrane protein biogenesis in bacteria,3 mitochondria,4, 5, 6, 7 and chloroplasts.8 More distantly-related YidC homologs (Get1, EMC3, and TMCO1) are found in the ER9, 10, 11 and also perform important roles in membrane protein insertion. All homologs in the family contain a conical-shaped 3-TMD core that contacts substrates for insertion, with YidC, Oxa1, & Alb3 containing additional transmembrane segments.12 In bacteria, the ER, and in chloroplast, the YidC/Oxa1 members can work together with the Sec translocase to insert proteins into the membrane.13, 14, 15 Independently of SecYEG, YidC/Oxa1 members insert membrane proteins with short translocated regions whereas the SecYEG translocase can insert transmembrane domains flanked by long translocated regions.16

A striking feature of the YidC protein (and all its homologs)17, 18, 19, 20, 21 is that it possesses a hydrophilic groove localized within the cytoplasmic leaflet of the membrane that is open to the membrane bilayer and the cytoplasm22, 23 (Figure 1A). Due to this structural feature, it has been assumed that YidC must use a different insertion mechanism than the Sec system, which contains a transmembrane (TM) aqueous channel that translocates substrates across the membrane through a centrally located pore ring and can shunt transmembrane segments sidewise into the membrane via a lateral gate region.24

The best studied substrate in terms of its YidC-mediated insertion mechanism is the Pf3 coat protein,25 a single-TM protein. The coat protein is known to insert in an N-out, C-in orientation and can insert post-translationally.26 Two models have been proposed for insertion of the Pf3 coat. One proposes that during membrane insertion by YidC, the substrate maintains a linear shape where the negatively charged periplasmic region of Pf3 coat first transiently interacts with the YidC hydrophilic groove, assisted by the strictly conserved positively charged residue in the groove, and then the hydrophobic segment of the substrate interacts hydrophobically with the TM segments of YidC to promote insertion. This was supported by findings of Kumazaki et al.22 The other model proposes that a hairpin loop structure is formed during YidC insertion where the substrate TM first moves up a “greasy slide” (TMs 3 and 5 of Gram negative YidC), and then the N-terminal periplasmic region transiently interacts with the YidC hydrophilic groove prior to translocating across the membrane.27

Our goal here was to examine a more complex substrate than Pf3 coat that is inserted by the YidC insertase. Therefore, we studied MscL, the large-conductance mechanosensitive channel (MscL),28, 29 that has two TM regions (Figure1A). It functions as a membrane-stretch-activated channel that responds to osmotic shock and typically has a homopentameric structure.30 Its TM1 shows an N-in, C-out orientation and TM2 has the inverse orientation, with a periplasmic region connecting them. Unlike Pf3, it is targeted to the membrane cotranslationally by the SRP pathway28 and is not dependent on the membrane potential for insertion.31 YidC is required for the membrane insertion of MscL,28, 29 although initially it was thought to play an essential role only in the assembly of the homopentamer.32 Further studies by Neugebauer et al. (2012) used cysteine crosslinking to show that I32 (TM1) and F85 (TM2) of MscL interact with YidC’s greasy slide at position 505 (TM5) and 430 (TM3)31 (Figure 1).

In this paper, we used a disulfide crosslinking approach to probe interactions between YidC and MscL during various stages of the membrane insertion process to elucidate MscL’s insertion mechanism. Our results demonstrate that the MscL TM1 and the TM2 segments are inserted into the YidC greasy slide co-translationally, showing a delayed interaction of TM1 and TM2 with the slide region. Using a cysteine modification method, we found that the periplasmic region only translocates after TM2 contacts the greasy slide, showing the pair of TM segments fully insert as a loop unit late in the synthesis of the protein. TM2 does not displace TM1 from the greasy slide during the insertion process as both TM segments remain in or near the YidC greasy slide region even when the full-length MscL is translated but still bound to the ribosome.