Figure 3. Posttranslational loss of ETC components in mtFAS mutants is specific to LYR proteins and their targets.

(A-B) Duplicate samples from the indicated cell lines were grown under proliferative conditions and subjected to TMT labeling and quantitative proteomics analysis. (A) Volcano plot of compiled Mecr clones vs. GFP controls showing all proteins (gray), mitochondrial proteins (blue), and electron transport chain subunits (ETC, red). Dashed gray lines indicate cutoffs for significance at -log10(p-value) = 1.3 and log2(Fold Change) = +/- 0.59. (B) Heatmap depicting log₂(Fold Change) of OXPHOS subunits in the indicated cell lines. (C) Quadruplicate samples from mtFAS mutant cells and controls were grown under proliferative conditions. Total RNA was isolated, used as input for mRNA library prep, and sequenced. Resulting data were aligned to the mouse genome and analyzed for differential expression. ETC subunit-encoding transcripts are shown in red vs. all other transcripts (gray). (D-G) Relative abundance of the indicated LYR proteins or their targets in the indicated cell lines from the quantitative proteomics experiment described in (A-B) #=p < 0.01, †=p < 0.001, ‡=p < 0.0001, error bars are SD. All statistical comparisons shown are between mtFAS mutants and GFP-1 clone; p-values when compared with GFP-2 clone were similar or smaller than when compared with GFP-1 clone. (H) Crude mitochondrial lysates generated from the indicated cell lines by differential centrifugation were normalized for total protein by BCA assay, separated by SDS-PAGE, and immunoblotted with the indicated antibodies.

Figure 3—figure supplement 1. Transcription of ETC subunit encoding genes and translation of mitochondrially encoded proteins are unaffected in mtFAS mutants.

(A-C) Quadruplicate samples from mtFAS mutant cells and controls were grown under proliferative conditions. Total RNA was isolated, used as input for mRNA library prep, and sequenced. Resulting data were aligned to the mouse genome and analyzed for differential expression. ETC subunit encoding transcripts are shown in red vs. all other transcripts (black). (C) Cumulative distribution plots showing no difference in abundance between ETC encoding transcripts vs. all other genes for the indicated mtFAS mutant vs. GFP control, p>0.05, Wilcoxon rank-sum test. (D) HEK293T cells were transiently transfected with human NDUFAB1-Flag or empty vector control along with the indicated LYRM-V5 constructs, grown for 48 hr after transfection, harvested, and crude mitochondrial lysates were prepared by differential centrifugation. Flag immunoprecipitation was performed and eluates were blotted with the indicated antibodies alongside 10% of input. (E-H) Duplicate samples from the indicated cell lines were grown under proliferative conditions and subjected to TMT labeling and quantitative proteomics analysis. Relative abundance of the indicated LYR protein (E), LYR target (F), mitochondrially encoded proteins (G), and MALSU1 (H). *=p < 0.05, #=p < 0.01, error bars are SD. All statistical comparisons shown are between mtFAS mutants and GFP-1 clone.