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The Assembly Pathway of Mitochondrial Respiratory Chain Complex I
Sergio Guerrero-Castillo, Fabian Baertling, Daniel Kownatzki, Hans J. Wessels, Susanne Arnold, Ulrich Brandt, Leo Nijtmans Cell Metabolism Volume 25, Issue 1, Pages (January 2017) DOI: /j.cmet Copyright © 2017 Elsevier Inc. Terms and Conditions
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Cell Metabolism 2017 25, 128-139DOI: (10.1016/j.cmet.2016.09.002)
Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure 1 Time Course of Subunit Recovery after Chloramphenicol Treatment The average relative abundance of N-module subunits NDUFV1, NDUFV2, and NDUFS1 was monitored. Mean values with SD from three independent complexome profiling datasets are given. (A) Total abundance of the subunits detected in all slices of the sample taken at the indicated time point relative to the control sample. (B) Cumulative abundance of assembled subunits migrating at >700 kDa relative to total abundance for all slices in the same sample. Cell Metabolism , DOI: ( /j.cmet ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure 2 Complexome Profiling Heatmaps of Complex I Subunits and Assembly Factors The migration profiles of complex I subunits and assembly factors were assembled manually and color coded by normalizing the relative abundance over control and all time points for each protein separately. Names of complex I subunits in black, assembly factors in red. See also Figure S1 and Tables S1 and S2. See Table S3 for an Excel version of the heatmaps. Cell Metabolism , DOI: ( /j.cmet ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure 3 Migration Profiles of Complex I Subassemblies
Segments of complexome profiles from selected samples are shown as heatmaps and plots to define the different assembly intermediates. The relative abundances were normalized to the maximum value within each segment. See Figure S1 for mass calibration. (A) 88 kDa, Q, and 170 kDa intermediates at 4 hr. (B) 67 kDa intermediate at 8 hr. (C) 72 kDa, 88 kDa, and N intermediates at 8 hr. (D) 293 kDa, 357 kDa, and PP-b intermediates at 24 hr. (E) 237 kDa and Q/PP-a intermediates in the control sample. (F) PD-b intermediate in the control sample. Cell Metabolism , DOI: ( /j.cmet ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure 4 Complex I Subassemblies and Subunits Persisting after 5 Days of Chloramphenicol Treatment The relative abundance of the average of the components of three small subassemblies (see Figure 3) and two unassembled subunits in the sample taken immediately after chloramphenicol removal (0 hr) relative to the control sample is shown. Mean values with SD from three independent complexome profiling datasets are given, except for NDUFAB1, which was only detectable in two of the samples in an amount sufficient for reliable label-free quantification. Cell Metabolism , DOI: ( /j.cmet ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure 5 Assembly Intermediates of Complex I Deduced from Complexome Profiling Analysis All components of the identified complexes are listed under the gel slice in which they were detected and arranged according to their assignment to the different submodules of complex I. The theoretical mass as calculated from the identified components and the name of the subassembly are indicated below. The molecular masses of the individual components are indicated on the right, taking into account the mass of the mature protein after cleavage of the mitochondrial targeting sequence where applicable (see Tables S1 and S2). The gel slice number and the interpolated molecular masses from both mass calibrations (for soluble and membrane proteins, respectively) are shown on top. However, interpolation of molecular masses must be taken with caution since several factors can influence protein migration in native electrophoresis gels. Assembly factors are shown in red and complexes III and IV in the supercomplex in pink. See also Figure S1, and for a detailed comparison with previous complex I assembly models see Table S4. Cell Metabolism , DOI: ( /j.cmet ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure 6 Supercomplex Formation
Formation of the assembled complexes was monitored by averaging the abundance values for all detected subunits. (A) Abundance heatmaps showing respiratory chain complexes I (CI), III (CIII), and IV (CIV) for the control sample and five time points after chloramphenicol removal. (B) Abundance profiles for the three respiratory chain complexes in the control sample. The insert shows the range of the supercomplexes from 1 to 3 MDa after normalization to the most abundant supercomplex S1 to illustrate the increasing relative abundance of complex IV from S0 to S2-4. (C–E) Change of relative abundance of respiratory chain complexes over time shown separately for the individual complexes (C), supercomplex III2-IV2 (D), and supercomplex S1 (E). Values were normalized to the control sample. Cell Metabolism , DOI: ( /j.cmet ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure 7 Comprehensive Model of Complex I Assembly
Cartoon representation of proposed assembly pathway of human mitochondrial complex I. Many subunit names were shortened by omitting the leading “NDUF.” For a detailed comparison with previous complex I assembly models see Table S4. Cell Metabolism , DOI: ( /j.cmet ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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