Fig. 3: In the absence of CLPP N-module is not exchanged on CI.
From: A salvage pathway maintains highly functional respiratory complex I
a De novo synthesis of mitochondrial proteins in wild type (+/+) and CLPP-deficient (−/−) MEFs followed by SDS-PAGE. Coomassie blue staining was used as a loading control. Individual lanes represent technical replicates of the wild type and CLPP-deficient MEF lines characterized by similar rates of mitochondrial translation. b Mitochondrial oxygen consumption rates (OCR; pmol O2/min) in wild type (+/+) and CLPP-deficient (−/−) MEFs and HeLa cells. Bars represent means ± SD. (MEFs n = 5–6; HeLa n = 6; biologically independent samples). Specific inhibitors used in the analysis are indicated. c Turnover of the CI subunits in wild type (+/+) and CLPP-deficient (−/−) MEFs. Cells were examined using pulsed SILAC complexome analysis after separation with BN-PAGE. Heatmap (top) shows protein abundance of previously synthesized proteins (light amino acids). Heatmap (bottom) shows newly biosynthesis and assembly of subunits in existing stable CI. Right panel represents the exchange rates of individual CI subunits on a fully assembled complex visualized on the CI structures. d, e Exchange rates of CI subunits in wild type (+/+) and CLPP-deficient (−/−) MEFs. Cells were examined using pulsed SILAC complexome analysis after separation with BN-PAGE. Bars represent mean ± SD. (**p < 0.01, ***p < 0.001). Paired Student’s t-test was used to determine the level of statistical difference. Detailed analysis is described in Supplementary Data 5. d Exchange rates of either all CI subunits (Assembl. CI) or only three (NDUFS1, NDUFV1, and NDUFV2) core N-module subunits (N-core) at the level of fully assembled CI (fractions 7628-995, n = 14) were calculated as ratio of newly synthesized to steady-state levels. e (left) Newly synthesized (new) and steady-state levels of core N-module subunits (NDUFS1, NDUFV1, and NDUFV2) at the level of free N-module subcomplexes (fractions 69-7, +/+ n = 12; −/− n = 15); (right) Exchange rates of core N-module subunits calculated as ratio of newly synthesized (red) to steady state (black) levels.
