Extended Data Figure 8 : Schematic explanation of β-galactosidase activity assay and MifM insertion activity.

From: Structural basis of Sec-independent membrane protein insertion by YidC

Extended Data Figure 8

a, b, MifM is a single-spanning membrane protein, and its membrane insertion is considered to be mediated by YidC (SpoIIIJ)21. To evaluate the MifM insertion activity of SpoIIIJ, we performed a genetic analysis using B. subtilis. In B. subtilis, SpoIIIJ is constitutively expressed, whereas YidC2 is expressed only when the SpoIIIJ activity is compromised, by the following mechanism. The expression of yidC2 is regulated by the upstream cis regulator open reading frame of mifM, which is co-transcribed with yidC2. During the synthesis of MifM, the C-terminal region of nascent MifM interacts with the peptide exit tunnel of the ribosome and causes translational arrest40,50. When the SpoIIIJ activity is normal, the translational arrest is released by the SpoIIIJ-dependent membrane insertion of MifM. Therefore, the translational arrest is transient or does not occur (a). By contrast, when SpoIIIJ activity is compromised, MifM is not inserted into the membrane, and its translation is arrested, which causes ribosome stalling. The stalled ribosome disrupts the downstream stem–loop structure and exposes the Shine–Dalgarno (SD) translation initiation signal sequence of the yidC2 messenger RNA (b). Thus, we can estimate the in vivo SpoIIIJ activity by measuring the expression of the introduced yidC2-lacZ fusion (Extended Data Fig. 7a): the reduction of MifM insertion efficiency by SpoIIIJ elevates the LacZ activity21,50.