Table S1 – Yeast strains and relevant genotype used in this work.

Table S2 – Plasmids constructed in this work.

Figure S1 – Alignments of yeast mitoribosome large subunit proteins with bacterial counterparts using ClustalW (https://www.ebi.ac.uk/Tools/msa/clustalo/) and box shade (https://embnet.vital-it.ch/software/BOX_form.html).

Table S3- Hydrogen bonds, Pi-stacking and Cation-Pi interactions of the mitochondria-specific extension from the studied proteins. The mitochondria-specific residues backbones (B), side-chains (SC), and interactors were visualized and annotated based on PDB- 5MRC structure.(https://www.rcsb.org/3d-view/5MRC/1)

Figure S2 – Mitoribosome structure based on PDB 5MRC – Light colours (light green, light yellow, light pink, light red) represent the mitochondria-specific extensions. Selected examples of annotated non-covalent interactions described in table S3. A) Tunnel exit components uL22, uL23 and uL24 are indicated together with associated proteins uL29 and bL17 – detailed interactions of uL22 K177 with mL44 D240; uL22 W291 with mL58 R188; Pi-stacking interaction of uL23 F226 with uL22 Y164; bL32 C120 and bL17 K150 in the coordination of a Zn ion. B) uL4, uL13, bL9, bL28 and uL30 are indicated – in detail: bL21 I88 – uL30 E80; uL13 Q147 – uL3 V27; bL9 S10 – 21S rRNA A1992; uL4 K278 –mL49 E69.

Figure S2 – Mitoribosome structure based on PDB 5MRC – Light colours represent the mitochondria-specific extensions (light green, light yellow, light pink, light red). Selected examples of annotated non-covalent interactions described in table S3 C) Central protuberance components uL5, uL16 and bL27; in detail: bL31 K130 – uS19 L21; bL27 H121 – uL5 H30; bL31 R27—21S rRNA A2426; uL5 H108 – uL16 Y204. D) uL3, bL19 and bL21; in detail: bL19 Y16 – 21SrRNA; uL3 V27 – uL13 Q147; bL21 N53 – mL58 V66.

Figure S3 – Growth properties of bL32 mutants. Differently of Fig 3 in the main text, here the YPEG plate was photographed after 7 days of incubation in order to register the poor growth of bL32-41 and bL32-42 mutants on non-fermentable carbon sources. The structures of the mentioned alleles are reproduced here.

Figure S4- Growth properties of bL9 mutants on ethanol-glycerol media (YPEG) is unchanged at 37oC