Figure 6.

Photoaffinity labeling of complex I by [¹²⁵I]IACS-010759-PD1. A, SMPs (4.0 mg of proteins/ml) were cross-linked by [¹²⁵I]IACS-010759-PD1 (10 nm), followed by the purification of complex I by BN-PAGE and electroelution. Isolated complex I was resolved by doubled SDS-PAGE, and the SDS gel was subjected to silver staining, autoradiography, or Western blotting using anti-bovine ND1 antibody. All data are representative of three independent experiments. B, localization of the region labeled by [¹²⁵I]IACS-010759-PD1. The labeled ND1 subunit was digested by Lys-C or Asp-N. The digests were resolved on a 16% Schägger-type SDS gel (16% T, 6% C, containing 6.0 m urea), followed by autoradiography. Proteins equivalent to ∼50 μg of SMPs were loaded into each well. All data are representative of three independent experiments. C, schematic representation of the digestion of ND1 by Lys-C or Asp-N. The TMHs were assigned according to the structures of bovine (13) or ovine (33) complex I. Predicted cleavage sites are denoted by arrowheads and marked with the residue numbers in the mature sequences of the bovine ND1 subunit (SwissProt entry P03887).