Fig 2: Schematic representation of human MIEF1 RefSeq variant 1 mRNA and altMiD51 and MiD51 proteins. A, Human MIEF1 includes 11 exons (RefSeq, GRCh38.p7). The mRNA variant 1 (NM_019008.5) shown here contains 6 exons. The CDS (blue) is shared between exons 3 to 6. AltMiD51 ORF is localized within exon 2, annotated as a non-coding exon. B, Sequence coverage in AP-MS experiments is represented in light colors. Proteotypic peptides sequence and positions (a.a.) are shown. EAVLSLYR and AISAPTSPTR peptides were selected for absolute quantification. *, known phosphorylated residue (phosphosite.org, (63)).

Fig 3: Src impacts on specific mitochondria-shaping proteins but not on mitochondrial fusion and fission rates. a Representative immunoblotting (n = 3–6) of the pro-fission proteins pS616-Drp1, pS637-Drp1, Drp1, FIS1, MiD49 and MiD51, of the pro-fusion proteins MFN1, MFN2 and OPA1, and of the total protein load (TPL) used as loading control, in Src+/+ and Src-/- MEFs. b, c Quantification of the immunoblotting shown for b pro-fission and c pro-fusion mitochondria-shaping proteins. Protein levels were normalized by TPL. d Quantification of mitochondrial fusion and fission events in live Src+/+ and Src-/- MEFs expressing mtGFP (n = 3) during 450 s videos. See also Movies 1 and 2. e Representative micrographs (n = 5) of Src+/+ and Src-/- MEFs expressing the mitochondria-targeted photo-activable GFP (mt-PAGFP) and mtDsRed after photoactivation of mt-PAGFP. f Quantification of GFP normalized to DsRed in region of interest (ROI) upon photoactivation as shown in e. g Live cell micrographs of Src+/+ and Src-/- MEFs expressing mtDsRed treated with FCCP (50 µM) as indicated. h Quantification of the number of cells with fragmented mitochondria as shown in g. Data are presented as mean ± S.E.M. A.U.: arbitrary unit. For b, c and d, *p < 0.05, **p < 0.01, according to Student’s t test. For f and h, data were analyzed with two-way ANOVA. Scale bars: 20 µm

Fig 4: DRP1 contributes to NADH- and succinate-linked respiration and mitochondrial ultrastructure formation.A–C, representative immunoblots and quantitation of DRP1, MFF, MiD49, MiD51, and OPA1 expression (N = 4 per condition). D–F, representative immunoblots and quantitation of PINK1, Parkin, and CI-V expression (N = 4 per condition). G, respiration supported by pyruvate and malate (PM), glutamate (G), and succinate (S) in the presence of ADP (P) or FCCP (E) in digitonin-permeabilized cells (N = 8 per condition). H, respiration supported by duroquinol (CIII) and ascorbate and TMPD (CIV) in the presence of ADP (P) or FCCP (E) in digitonin-permeabilized cells (N = 8 per condition). I, enzymatic activity of citrate synthase (N = 10 per condition). J, mtDNA content (COX2:18S) (N = 6 per condition). K, representative transmission electron microscopy (TEM) of phosphate-buffered aldehyde-fixed cells (500 nm). L–N, quantitation of mitochondrial branching and complexity (form factor), roundness, and length (aspect ratio) (shCTRL: 56 mitochondria, shDRP1: 43 mitochondria). B, C, E, F, I, and J are displayed as a box (mean ± 5–95% confidence interval) and whiskers (minimum to maximum) plot. G and H are displayed as the mean ± SEM with all points. L–N are displayed as a violin plot. *p < 0.05, **p < 0.01, ***p < 0.01, ****p < 0.001. B, C, E, F, I, and J were assessed by unpaired Student’s t test. G and H were assessed by one-way ANOVA with Tukey’s multiple comparisons. L–N were assessed by Mann–Whitney U test.

Fig 5: Mitochondrial matrix-localized Src modulates mitochondrial size independently of Drp1, MiD51, OPA1 and cellular respiration. a Representative immunoblotting (n = 3) of Src, Drp1, MiD51, OPA1 and total protein load (TPL), used as loading control, in Src+/+ and Src-/- MEFs expressing pcDNA, Src-HA and mtSrc-HA. b Quantification of immunoblotting shown in e. Protein levels were normalized by TPL. c Cellular respiration of Src+/+ and Src-/- MEFs expressing pcDNA, Src-HA and mtSrc-HA (n = 3). Data with different letters are statistically different (p < 0.05), according to one-way ANOVA (for b) or two-way ANOVA (for c) followed by Tukey’s post hoc test. A.U. arbitrary unit