Figure 2.

YidC makes multiple contacts to SecY through its transmembrane and periplasmic domains. In vivo photo cross-linking was performed as described in Fig. 1 using the co-expression system with YidC variants containing pBpa at the indicated positions. After UV-exposure, samples were purified via the C-terminal His-tag on SecY and analysed on western blot with α-YidC antibodies (a–c). The 95 kDa YidC-SecY cross-links are indicated. For position D236, an additional strong band below the 130 kDa was recognized by α-YidC antibodies (*). In addition, α-YidC antibodies recognized a UV-dependent band at approx. 65 kDa (#). (d) In vitro photo cross-linking was performed using inner membrane vesicles carrying 10 μg of wild type YidC or YidC-pBpa variants co-expressed with SecY_HisEG. Samples were purified by metal affinity chromatography using the His-tag on SecY and subsequently analysed on western blot with α-YidC antibodies. Uncropped images are displayed in Supplementary Figure S4.