Fig. 6.

Analysis of ND2 mutations in nuoN of E. coli. (A) The locations of human mutations F60S (red) and L71P (blue) are shown in ND5 (light blue), using PDB file 5xtd (Gu et al., 2016). (B) ND4L (violet) is shown. (C) The locations of E. coli residues T160 (red) and L171 are shown in nuoN (light blue), using PDB file 3rko (Efremov and Sazanov, 2011). (D) nuoK (violet) is shown. (E, F) dNADH-oxidase activities of membrane vesicles prepared from the E. coli mutants are shown compared to a wild type sample prepared the same day. (G, H) Proton translocation rates from the same samples shown in panels E and F are indicated by fluorescence quenching of the acridine dye ACMA. (I) Membrane vesicles from E. coli cells carrying the modeled human mutations, nuoN_T160F and nuoN_L171P, in pBAD33-(I-N), were prepared and solubilized with dodecyl maltoside. The samples, including a control expressing the entire nuo operon, pBAD33-(A-N), were immunoprecipitated with an HA antibody to bring down the HA-tagged subunit N. The input panel shows the presence of subunit K in all samples, while the IgG panel shows that in the absence of HA-antibody, no K subunits were precipitated. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)