Nature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1. Generation and validation of mtef4-knockout mice.

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1 Supplementary Figure 1 Generation and validation of mtef4-knockout mice. (a) Alignment of EF4 (E. coli) with mouse, yeast and human EF4. (b) Domain structures of mouse mtef4 compared to those of EF4 (E. coli). (c) mtef4 is ubiquitous in mice tissues and organs. (d) Schematic strategy for the generation of mtef4 KO mouse. (e) Southern blot verification of a single introduction of the targeting construct in the homologous recombined embryonic stem (ES) cell clones. C: control, 129 ES cells. 1-3: positive clones. 5 -: 5 -primer. 3 -: 3 -primer. (f) PCR-based genotyping of KO and gko mice. (g) qpcr of mtef4 transcripts from various tissues of WT and KO mice (mean ± s.d., n = 12 mice ). (h) Western blotting (WB) of mtef4 protein from total WT (+/+) and KO (-/-) mice, and gko mice.

2 Supplementary Figure 2 Morphology of mitochondria-rich tissues and hormones from WT and total-ko male mice. (a, b) Macrograph (a) and Hematoxylin and eosin (H&E) staining (b) of the heart. Scale bar: 200 μm (c) H&E staining of the liver, muscle and cerebrum. Scale bar: 200 μm (d) Quantification of the seminiferous tubule diameters (mean ± s.d., n = 5 mice, **P < 0.01). (e-g) Hormone changes upon mtef4 ablation. Comparison of WT, KO and gko mice serum follicle-stimulating hormone (FSH) (e), luteinizing hormone (LH) (f) and testosterone (T) (g) (mean ± s.d., n = 5 mice, *P < 0.05, **P < 0.01).

3 Supplementary Figure 3 Morphology of mitochondria-rich tissues from WT and gko male mice. (a-c) H&E staining of the heart, liver, muscle, cerebrum (a), testis (b) and epididymis (epi) (c) from WT and gko mice. Scale bar: 200 μm. Red frame: zoom-in, scale bar: 50 μm. Red circle: immature spermatogenic cells. (d) The concentration of the spermatozoa in the epididymis. (e) Morphology of the sperms. Hoechst33258 (blue) and mitotracker (red) staining indicates nuclei and mitochondria, respectively. Abnormal mitochondrial sheaths are indicated by white arrows. Scale bar: 50 μm. (f, g) The mobility of the sperms. (h) Computer-assisted sperm analysis (CASA) parameter of the sperms. Average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), beat cross frequency (BCF) (mean ± s.d., n = 10 mice, ***P < 0.001). (i, j) EF4 is co-localized with the ribosome in the mitochondria of mouse testicular tissue. (k, l) Transmission electron microscope (TEM) photographs of gko testis (k) and the sperms (l). Mt: mitochondria, Nu: nucleus, scale bar: 1 μm. Red frame: zoomin. Scale bar: 1 μm.

4 Supplementary Figure 4 Qualitative and quantitative analysis of OXPHOS subunits from mitochondria-rich tissues of WT and KO male mice. (a) Blue native gels (BNG) of heart, liver, muscle and cerebrum mitochondria. (b) In-gel activity (IGA) of complex I and IV. (c) Quantification of IGA from complex I (left) and complex IV (right) (mean ± s.d., n = 4 mice). (d) WB of nuclear and mitochondrial DNA encoded OXPHOS complex subunits. (e) Relative amount (%) of arbitrary units from samples in (d). Tom20: internal control, mean ± s.d., n = 5 mice.

5 Supplementary Figure 5 Qualitative and quantitative analysis of OXPHOS complexes and subunits from mitochondria-rich tissues of WT and gko male mice. (a, b) BNG (a) and IGA (b) of heart, liver, muscle and cerebrum mitochondria show similar amount of OXPHOS complexes in WT and gko mice. (c) Quantification of IGA from complex I (left) and complex IV (right) (mean ± s.d., n = 4 mice). (d) IGA of OXPHOS complexes from WT and gko heart, testis and epididymis. (e, f) WB of ndna (e) and mtdna (f) encoded OXPHOS complex subunits in the samples as in (d). (g, h) Relative amount (%) of arbitrary units from samples in (e) and (f), respectively. H: heart, T: tesits, E: epididymis, Tom20: internal control, mean ± s.d., n = 5 mice, **P < 0.01, ***P <

6 Supplementary Figure 6 Quantification of mtdna, mrna of OXPHOS subunits, and mitochondrial ribosomes. (a) Relative mtdna content (mean ± s.d., n = 10 mice, *P < 0.05). (b) Relative quantification of ndna (blue) and mtdna (red) encoded OXPHOS subunits. The ratio was defined as the qpcr value of each tested transcript from KO sample versus from WT sample. Actin: internal control, mean ± s.d., n = 10 mice. (c) Quantification of the expression of mitochondrial transcription factor TFAM (mean ± s.d., n = 5 mice, *P < 0.05). (d) Quantification of the expression of mitochondrial small and large ribosomal subunit protein, MRPS18 and MRPL11, respectively. Tom20: internal control, mean ± s.d., n = 3 mice.

7 Supplementary Figure 7 [ 35 S]methionine pulse-chase-labeling and protein synthesis of isolated mitochondria from WT and gko testes. Purified mitochondria were pulse labeled with [ 35 S]-Met for 1 h and subsequently chased by the addition of an excess of unlabeled Met with 3 h and 5 h incubation in the presence (a) or absence (c) of protease inhibitor. The thirteen mitochondrial translation products are indicated. (b, d) The quantification of (a) and (c), respectively. Internal control: Tom20, mean ± s.d., n = 3 mice, ***P <

8 Supplementary Figure 8 mtor inhibition in the heart results in OXPHOS defects, and deletion of mtef4 induces downregulation of cytoplasmic translation in sperm cells. (a) Wet weight of the hearts from male mice treated with DMSO or rapamycin (mean ± s.d., n = 5 mice, **P < 0.01). (b) Quantification of the OXPHOS subunits of the DMSO/rapamycin treated mice. The quantitative value of the sample WT heart treated with DMSO was defined as 100%. Tom20: internal control, mean ± s.d., n = 3 mice, *P < 0.05, **P < (c, d) Cytoplasmic polysome patterns of WT (blue) and gko (red) tissues. On the right, the monosomes (80S) versus polysomes ratio was quantified (mean ± s.d., n = 3 mice, *P<0.05). (e, f) Distribution of ATP5D mrnas across the density gradients from (c, d) was determined by RT-sqPCR. Red square, increased subunits and monosomes in gko testis than in WT testis.

9 Supplementary Table 1. Average weight of mito-rich organs from WT and KO. Organ Weight (mg) +/+ -/- Body Weight (g) 41.5± ±1.7 Cerebrum 452±21 473±11 Heart 151±20 167±12 Liver(g) 1.65± ±0.30 Testis 240±10 127±16*** Epididymis 77±6 66±11 mean ± s.d., n = 10 mice, ***P < Supplementary Table 2. Average weight of mito-rich organs from WT and gko. Organ Weight (mg) +/+ -/- Body Weight (g) 41.9± ±3.3 Cerebrum 442±30 443±31 Heart 130±20 112±20 Liver(g) 1.33± ±0.17 Testis 222±24 129±25*** Epididymis 79±3 69±2 mean ± s.d., n = 10 mice, ***P <