Bioinformatic analysis classifies the human protein encoded by immature colon carcinoma transcript-1 (ICT1) as one of a family of four putative mitochondrial translation release factors. However, this has not been supported by any experimental evidence. As only a single member of this family, mtRF1a, is required to terminate the synthesis of all 13 mitochondrially encoded polypeptides, the true physiological function of ICT1 was unclear. Here, we report that ICT1 is an essential mitochondrial protein, but unlike the other family members that are matrix-soluble, ICT1 has become an integral component of the human mitoribosome. Release-factor assays show that although ICT1 has retained its ribosome-dependent PTH activity, this is codon-independent; consistent with its loss of both domains that promote codon recognition in class-I release factors. Mutation of the GGQ domain common to ribosome-dependent PTHs causes a loss of activity in vitro and, crucially, a loss of cell viability, in vivo. We suggest that ICT1 may be essential for hydrolysis of prematurely terminated peptidyl-tRNA moieties in stalled mitoribosomes.

Human mitochondria are not strict adherents to the universal genetic code, with modifications that include the apparent recoding of two arginine triplets to termination signals. This use of both AGA and AGG occurs rarely in other mammals, and this putative change has long posed a challenging conundrum. A -1 mitoribosome frameshift upstream of the rare codons would necessitate recognition of only the conventional UAA and UAG termination codons. By using a sequence-specific endoribonuclease, we show that the rare arginine codons, presumably in association with other cis elements, promote frameshifting in human mitoribosomes.

Mutations in the AFG3L2 gene have been linked to spinocerebellar ataxia type 28 and spastic ataxia-neuropathy syndrome in humans; however, the pathogenic mechanism is still unclear. AFG3L2 encodes a subunit of the mitochondrial m-AAA protease, previously implicated in quality control of misfolded inner mitochondrial membrane proteins and in regulatory functions via processing of specific substrates. Here, we used a conditional Afg3l2 mouse model that allows restricted deletion of the gene in Purkinje cells (PCs) to shed light on the pathogenic cascade in the neurons mainly affected in the human diseases. We demonstrate a cell-autonomous requirement of AFG3L2 for survival of PCs. Examination of PCs prior to neurodegeneration revealed fragmentation and altered distribution of mitochondria in the dendritic tree, indicating that abnormal mitochondrial dynamics is an early event in the pathogenic process. Moreover, PCs displayed features pointing to defects in mitochondrially encoded respiratory chain subunits at early stages. To unravel the underlying mechanism, we examined a constitutive knockout of Afg3l2, which revealed a decreased rate of mitochondrial protein synthesis associated with impaired mitochondrial ribosome assembly. We therefore propose that defective mitochondrial protein synthesis, leading to early-onset fragmentation of the mitochondrial network, is a central causative factor in AFG3L2-related neurodegeneration. IntroductionEukaryotic cells monitor the quality of their mitochondria using mechanisms acting at the interorganelle or intraorganelle level. Mitochondrial fusion and fission ensure maintenance of a functional network, whereas intramitochondrial proteases remove misfolded and aggregated proteins and also have a role in regulating mitochondrial function by processing specific substrates (1). Both levels of quality control are crucial in neurons, as emphasized by the increasing number of neurodegenerative diseases linked to impaired mitochondrial dynamics or mutations in mitochondrial proteases (1).Subunits of the matrix ATPase associated with various cellular activities (m-AAA) protease have emerged as crucial players in defending neurons against neurodegeneration. The m-AAA protease is an evolutionary conserved hexameric complex in the inner mitochondrial membrane, exposing the catalytic domains to the matrix (2, 3). Studies in yeast and mammals have shown that the m-AAA protease degrades misfolded polypeptides and excess protein components lacking assembly partners in the inner mitochondrial membrane (2,4,5). Moreover, the yeast m-AAA protease mediates proteolytic maturation of the mitochondrial ribosomal component MRPL32 (6). MRPL32 maturation is a prerequisite for mitochondrial translation and the synthesis of mitochondrially encoded (mt-encoded) respiratory chain subunits, explaining respiratory deficiencies of yeast cells lacking the m-AAA protease (6). In a reconstituted yeast system, the mammalian m-AAA protease was able to process MRPL32, suggesting evolutionary conservation of this...