FIGURE 8.

Cross-linking between the M and N subunits within the membrane. A, side view of the membrane arm of Complex I showing the sites of the two cross-links within the membrane. Subunit L is colored blue, subunit M is magenta, subunit N is green, and subunits A, J, and K are colored gray. Met-145 and Ala-155 (both TM5) are from subunit M, and Met-388 (TM12b) and Val-430 (TM13) are in subunit N. The image was developed from Protein Data Bank code 3rko (16). B, representative immunoblots of membrane samples from M145C (nuoM) + M388C (nuoN) and A155C (nuoM) + V430C (nuoN) are shown with and without treatment with the MTS reagent M2M to form cross-links. All blots are probed with antibody to subunit M, which does not appear to recognize the cross-linked products. C, the effect of treatment with M2M on the deamino-NADH oxidase activity of the mutants is shown. Results are the means and S.E. of at least four measurements from at least two membrane preparations. D, proton translocation assays of the mutants with and without treatment with M2M are shown. The fluorescence quenching of ACMA was initiated by deamino-NADH and was reversed by the addition of FCCP.