CLPP coordinates mitoribosomal assembly through the regulation of ERAL1 levels
Despite being one of the most studied proteases in bacteria, very little is known about the role of ClpXP in mitochondria. We now present evidence that mammalian CLPP has an essential role in determining the rate of mitochondrial protein synthesis by regulating the level of mitoribosome assembly. Through a proteomic approach and the use of a catalytically inactive CLPP, we produced the first comprehensive list of possible mammalian ClpXP substrates involved in the regulation of mitochondrial translation, oxidative phosphorylation, and a number of metabolic pathways. We further show that the defect in mitoribosomal assembly is a consequence of the accumulation of ERAL1, a putative 12S rRNA chaperone, and novel ClpXP substrate. The presented data suggest that the timely removal of ERAL1 from the small ribosomal subunit is essential for the efficient maturation of the mitoribosome and a normal rate of mitochondrial translation.To determine the in vivo function of CLPP in mammals, we generated a conditional knockout allele of the mouse Clpp gene (Fig 1A and B). CLPP‐deficient mice (Clpp −/− ) were obtained from intercrossing of Clpp +/− animals (Fig 1C), and the loss of CLPP was confirmed on both transcript and protein level (Fig 1D and E). Although we regularly acquired Clpp −/− mice, they were not born in Mendelian ratios (Table EV1), likely dying in utero, since no postnatal lethality prior to the age of weaning was observed. Currently, we follow an aging cohort of CLPP‐deficient mice that show the same survival as respective wild‐type littermates at least up to 2 years of age. This is in contrast to findings on a similar mouse model reported to have increased postnatal mortality and possibly shorter life span, although we also observed general sterility of both CLPP‐deficient genders and shorter stature of the mice, as describe earlier (Gispert et al, 2013). Besides differences in the genetic approach used to generate CLPP‐deficient mice (conditional knockout targeting versus gene‐trap technology), also different hygienic level in animal facilities might contribute to these disparities. Overall, our results suggest that CLPP function is essential in some critical period during development and that in the postnatal life mice can tolerate a CLPP deficiency.Clpp gene targeting was carried out at Taconic Artemis, Germany, in Art B6/3.5 embryonic stem cell line on a genetic background of C57BL/6 NTac. LoxP sites flanked exons 3–5 of the Clpp gene. A puromycin resistance selection marker was inserted into intron 5 and flanked by F3 sites. The targeted locus was transmitted through germline that resulted in heterozygous Clpp +/PuroR−loxP mice. Animals were bred with B6 Flp_deleter transgenic (TG) mice to remove the puromycin selection marker. Finally, Clpp +/loxP mice were mated with mice expressing Cre recombinase under the promoter of beta‐actin resulting in deletion of exons 3–5. This deletion resulted in loss of function of the Clpp gene, by removing a part of the protease domain and generated a frameshift from exon 2–6. Clpp +/− heterozygous mice were intercrossed to obtain the Clpp −/− homozygous knockout mice. The genotyping primers used are:Animal experiments were in accordance with guidelines for humane treatment of animals and were reviewed and approved by the Animal Ethics Committee of North‐Rheine Westphalia, Germany.The work was supported by grants of the European Research Council (ERC‐StG‐2012‐310700), and German Research Council (DFG). P.M and C.B. received scholarships from CECAD graduate school.