Fig 2: Biological pathways impacted by the loss of ECHS1 protein. (A) Volcano plot of 274 differentially expressed proteins from isolated ECHS1 KO mitochondria; 142 are upregulated and 132 are downregulated compared to control. ECHS1 is shown in red in the top left quadrant. Permutation-based FDR set at < 1% and s0 = 1. ECHS1 was present in control (CON) cells but at negligible levels in ECHS1 KO cells. Significantly altered proteins which also had significantly altered transcript levels are numbered in red. (B) Functional enrichment of mitochondrial proteins using GO terms and KEGG pathways. Shown are top tier terms with Fisher's exact test and Bonferroni post hoc testing with P < 0.05. (C) Significantly decreased proteins in ECHS1 KO mitochondria that also had significantly reduced transcript levels detected by RNA-seq (proteins 1–16 in Volcano plot, ranked by protein log2 fold change). (D) Significantly increased proteins in ECHS1 KO mitochondria that also had increased transcript levels detected by RNA-seq (proteins 17–21 in Volcano plot, ranked by protein log2 fold change).

Fig 3: Downregulated mitochondrial metabolic pathways in ECHS1 KO cells. (A) Volcano plot of Reactome gene set enrichment. Out of 1472 gene sets analysed, 43 were upregulated and 464 were downregulated in ECHS1 KO cells. Red points indicate FDR < 0.05. (B) Mitch pathway analysis indicating top 20 differentially expressed gene sets as ranked by effect size in ECHS1 KO cells. (C) Ridge plot of 10 key gene sets that are differentially expressed in ECHS1 KO cells (FDR < 0.05).

Fig 4: ECHS1 KO cells have decreased OXPHOS protein steady-state levels. (A) Steady-state levels of NDUFB8 and MT-CO2 were decreased in ECHS1 KO mitochondria, whereas levels of the voltage-dependent anion-selective channel protein 1 (VDAC1) were increased. (B) Steady-state levels of complex I (CI), the complex III dimer (CIII2), complex IV (CIV), complex V (CV), the CIII2/CIV supercomplex and the CI/CIII2/CIV supercomplex were all reduced in ECHS1 KO mitochondria compared to control (CON) mitochondria. Data shown as mean ± SD, n = 3. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to control values (Student's two-tailed t-test).

Fig 5: Co-immunoprecipitation of ECHS1 identifies putative OXPHOS complex I binding partners, with loss of ECHS1 disrupting complex I assembly. Co-immunoprecipitation with ECHS1 antibodies (ECHS1-PAS) or PAS alone, followed by western blotting and immunodetection with anti-ECHS1 antibodies. (A) ECHS1-PAS pull-down captured ECHS1 (Lane 1) from control (CON) mitochondria, with non-specific proteins also pulled down (lane 1). ECHS1 was not captured from ECHS1 KO mitochondria as expected, however non-specific proteins were pulled down (lane 2). PAS alone did not capture any non-specific proteins detectable with anti-ECHS1 antibodies (Lanes 3 and 4) from either CON or ECHS1 KO mitochondria. Image shown is representative of three independent experiments. (B) Six proteins specific for ECHS1 immunocapture identified by comparative analyses of co-immunoprecipitated proteins captured from CON mitochondria with those captured from ECHS1 KO mitochondria. Of these, RNA-seq identified reduced transcript levels of NDUFB11, NDUFV3 and RPL12 (ns, not significant). (C) Volcano plot of captured proteins by ECHS1 coimmunoprecipitation. Permutation-based FDR set at < 1% and s0 = 1. RPL12, RPL29, NDUFB11, NDUFV3, ATP5D and ACADVL (shown in red) present in capture from CON mitochondria but not ECHS1 KO mitochondria (n = 3, Student's two-tailed t-test, P < 0.05). (D) BN-PAGE showing the assembly of NDUFA9 into mature complex I (CI) following solubilisation of 40 µg mitochondria per lane in 1% TX-100. (E) SDS/PAGE showing NDUFA9 following import into both CON and ECHS1 KO mitochondria. NDUFA9 is detectable in its precursor (p) form and as a proteinase K (PK+) resistant mature (m) form. (F) Quantitation of NDUFA9 assembly into mature complex I after 10, 30 and 60 min of import, normalised to maximum amount of NDUFA9 imported after 60 min in CON. The amount of NDUFA9 assembled into complex I (CI) after 60 min in ECHS1 KO mitochondria was only 37.9 ± 19.3% of CON levels. Data shown are mean ± SD with n = 3, **P < 0.01 relative to control (CON; Student's two-tailed t-test). Images shown are representative of three independent experiments.