Mitochondria are primary ATP-producing organelles which originated from an ancestral alpha-proteobacterial endosymbiont (1
). The oxidative phosphorylation (OXPHOS) pathway, consisting of the mitochondrial respiratory chain and the ATP synthase complex, is the final biochemical pathway in energy conversion (3
). However, mitochondria have been found to perform pivotal roles in many other metabolic, regulatory and developmental processes. During the evolution of the mitochondria, most genes of the ancestral endosymbiont have been transferred from the mitochondrial to the nuclear genome. The genes that reside on the mitochondrial DNA (mtDNA), e.g. 13 OXPHOS-related genes in human, are translated into their respective protein products by the mitochondrial translation machinery, which resembles the prokaryotic translation system (4
), comprising a mitochondrial ribosome (mitoribosome) and several translation factors. In mammals, all proteins that constitute the mitochondrial translation machinery are encoded by the nuclear genome. Also the mitochondrial genomes of fungi generally encode only very few mitochondrial ribosomal proteins (MRPs). In contrast, the mitochondrial genome of many protozoa and plants encode numerous MRPs. Most notably, the ‘primitive’ mitochondrial genome of the freshwater protist Reclinomonas americana
was found to contain as many as 27 MRP-encoding genes (5
Mitoribosomes have undergone major remodeling during their evolution. For instance, it has been found that, despite the fact that their rRNA content is only half the content of bacterial ribosomes (6
), mitoribosomes generally exceed the bacterial ribosomes both in molecular mass as well as in physical dimensions (6–8
). Mass spectrometry studies of the bovine and yeast mitoribosomes, which have served as model systems during the past years, revealed that mammalian mitoribosomes comprise about 80 MRPs (9–13
). In yeast, about 70 MRPs have been identified thus far, although protein–protein interaction data and mutational analysis suggest that this number might be substantially higher (14–16
). Interestingly, a recent proteomics survey of mitochondrial ribosome-related complexes in the kinetoplast-mitochondria of Leishmania tarentolae
revealed an additional protein complex which was found to be specifically associated with the small subunit (SSU), but not with the large subunit (LSU) of the mitoribosome (17
). The biological role of the additional SSU-associated complex, which mostly consists of proteins unique to kinetoplastida, remains unknown.
Apparently, mitoribosomes have expanded their protein content in the course of evolution by acquiring numerous extra ‘supernumerary’ MRPs. Currently information regarding the functions of these supernumerary MRPs is rather limited. In addition, loss of MRPs that are normally part of the ‘bacterial core ribosome’ is also observed.
Recently, several attempts have been made to identify the MRPs in other eukaryotes, such as Neurospora crassa
), Arabidopsis thaliana
) and L. tarentolae
), however, a comprehensive study of the evolution of the mitoribosomal proteome remains to be established. Such a study bears relevance for the potential identification of genes that cause mitochondrial dysfunctions in human patients. Since mitochondria perform many fundamental functions, mitochondrial dysfunction results in a wide variety of multisystemic diseases, predominantly affecting tissues with high metabolic energy rates (21
). These disorders are mostly caused by the dysfunction of one or more enzyme complexes of the OXPHOS system and several mutations have been identified in mtDNA (22
) as well as in nuclear DNA (23
). Not only mutations in structural OXPHOS genes, but also mutations in genes involved in the mitochondrial transcription or translation, or in the assembly of OXPHOS complexes can result in OXPHOS disease. Although the vast majority of components of the mitochondrial translation system are nuclear encoded, thus far most mutations associated with mitochondrial translation defects have been reported in mtDNA-encoded tRNAs and rRNAs (22
). Recently, mutations in four different nuclear gene products involved in mitochondrial protein synthesis have been reported in patients with mitochondrial disease: elongation factors EFG1 (25
), EFTs (26
) and EFTu (27
), and small ribosomal subunit protein MRPS16 (28
). Additional information regarding which MRPs could be implicated in disease could be obtained by studying the evolutionary conservation of the MRPs.
The aim of the present study is 2-fold, from an evolutionary and a disease point of view: (i) gain insight into the evolution of the mitoribosome and its protein content in various eukaryotic species; (ii) prioritize MRPs as candidates for their involvement in mitochondrial disease. We have performed a comprehensive comparative genomics analysis of the MRPs in 18 eukaryotic species from which both the complete nuclear and the organellar genome sequences were available. Representatives are included from different phylogenetic groups: six metazoa (Homo sapiens, Mus musculus, Tetraodon nigroviridis, Drosophila melanogaster, Anopheles gambiae and Caenorhabditis elegans), two fungi (Saccharomyces cerevisiae and N. crassa), one microsporidian (Encephalitozoon cuniculi), one mycetozoan (Dictystelium discoideum), one plant (A. thaliana), one alga (Chlamydomonas reinhardtii), one apicomplexa (Plasmodium falciparum), one ciliophora (Tetrahymena thermophila), two kinetoplastida (Trypanosoma brucei and Leishmania major), one diplomonad (Giardia lamblia) and the mitochondrial genome of the freshwater protist Reclinomonas americana. In addition, we included a balanced, phylogenetically diverse subset of completely sequenced prokaryotic genomes in our survey.
Our analysis retrieved multiple previously unidentified MRPs in several species, two of which constitute potential novel human MRPs. Furthermore, the current study established orthology relationships between seven MRPs reported to be fungi specific and ribosomal proteins from other eukaryotes. The establishment of these homology relationships enabled us to trace the origins of some of the supernumerary MRPs and to predict their molecular functions. In addition, we have investigated all MRPs in the presence of additional, newly acquired protein domains that might point at mitochondria-specific adaptations of the mitoribosome, and we have reconstructed the evolutionary history of the mitoribosomal proteome in terms of gains and losses of the MRPs along the different eukaryotic lineages. The newly detected mitochondrial ribosomal genes constitute, in addition to the set of evolutionary conserved MRPs, excellent new screening targets for human patients with unresolved mitochondrial oxidative phosphorylation disorders.