MitoRibo-Tag Mice Provide a Tool for In Vivo Studies of Mitoribosome Composition

Mitochondria harbor specialized ribosomes (mitoribosomes) necessary for the synthesis of key membrane proteins of the oxidative phosphorylation (OXPHOS) machinery located in the mitochondrial inner membrane. To date, no animal model exists to study mitoribosome composition and mitochondrial translation coordination in mammals in vivo. Here, we create MitoRibo-Tag mice as a tool enabling affinity purification and proteomics analyses of mitoribosomes and their interactome in different tissues. We also define the composition of an assembly intermediate formed in the absence of MTERF4, necessary for a late step in mitoribosomal biogenesis. We identify the orphan protein PUSL1, which interacts with a large subunit assembly intermediate, and demonstrate that it is an inner-membrane-associated mitochondrial matrix protein required for efficient mitochondrial translation. This work establishes MitoRibo-Tag mice as a powerful tool to study mitoribosomes in vivo, enabling future studies on the mitoribosome interactome under different physiological states, as well as in disease and aging. [Display omitted] •MitoRibo-Tag mice with a tag on mL62 were generated to study mitoribosomes in vivo•The mitoribosome interactome of different mouse tissues was defined with proteomics•PUSL1 was identified as a mitoribosome-interacting protein using MitoRibo-Tag mice•MitoRibo-Tag mice allow mitoribosome analysis under different conditions and setups Busch et al. generated MitoRibo-Tag mice to study mitoribosome composition in vivo in different tissues. Proteomics of MitoRibo-Tag mice with defective assembly identified PUSL1 as a mitoribosome-interacting protein required for efficient mitochondrial translation. MitoRibo-Tag mice provide a tool to study mitoribosomes under different physiological conditions and in disease and aging.