FIGURE 1.

In vitro photocross-linking of YidC to the signal peptide of nascent Hbp. A, schematic representation of nascent 116HbpTAG41. The classical region of the Hbp signal peptide is indicated as a thick solid line with a white central area representing the hydrophobic core. A signal peptide extension that is conserved among a subset of ATs is indicated by a hatched box. The position of the amber codon that is suppressed with (Tmd)Phe-tRNA^sup to allow incorporation of a photoreactive probe is indicated (TAG41). The nascent chain is depicted with 35 amino acids in the ribosome. B, in vitro translation of 116HbpTAG41 in the presence of inverted inner membrane vesicles. After translation, samples were irradiated with UV light to induce cross-linking or left in the dark as indicated. C, UV-irradiated samples described under B were subjected to extraction with sodium carbonate (Na₂CO₃). The resulting carbonate pellet and supernatant (sup.) fractions were analyzed by SDS-PAGE directly, or first immunoprecipitated (IP) using antiserum against YidC, SecY, TF, or fifty-four homologue as indicated. Adducts representing cross-linking to either SecY or fifty-four homologue are indicated with an caret. Molecular mass (kDa) markers are indicated at the left side of the panels.