Reconstructing the evolution of the mitochondrial ribosomal proteome

Abstract

For production of proteins that are encoded by the mitochondrial genome, mitochondria rely on their own mitochondrial translation system, with the mitoribosome as its central component. Using extensive homology searches, we have reconstructed the evolutionary history of the mitoribosomal proteome that is encoded by a diverse subset of eukaryotic genomes, revealing an ancestral ribosome of alpha-proteobacterial descent that more than doubled its protein content in most eukaryotic lineages. We observe large variations in the protein content of mitoribosomes between different eukaryotes, with mammalian mitoribosomes sharing only 74 and 43% of its proteins with yeast and Leishmania mitoribosomes, respectively. We detected many previously unidentified mitochondrial ribosomal proteins (MRPs) and found that several have increased in size compared to their bacterial ancestral counterparts by addition of functional domains. Several new MRPs have originated via duplication of existing MRPs as well as by recruitment from outside of the mitoribosomal proteome. Using sensitive profile-profile homology searches, we found hitherto undetected homology between bacterial and eukaryotic ribosomal proteins, as well as between fungal and mammalian ribosomal proteins, detecting two novel human MRPs. These newly detected MRPs constitute, along with evolutionary conserved MRPs, excellent new screening targets for human patients with unresolved mitochondrial oxidative phosphorylation disorders.

Figure 1.

Distant homology between mitochondrial and bacterial ribosomal protein families detected in this study. Multiple sequence alignments of MRPL47 and RPL29 proteins (A) and of MRPS24 and RPS3 proteins (B). Amino acid residues are shaded according to the following physicochemical properties: t: tiny (blue font on yellow shading); m: amphoteric (red font on green shading); h: hydrophobic (white font on black shading); o: positive (red font on blue shading); p: polar (black font on green shading); s: small (green font on yellow shading); a: aliphatic (red font on gray shading), r: aromatic (blue font on gray shading); c: charged (white font on blue shading). Sequences are annotated by the organism name and Swiss-Prot protein identifier and the boundaries of the segment that is used in the alignment are indicated. A 90% consensus sequence is depicted below the alignments using the same classification, with invariant residues being indicated in capitals.

Figure 2.

Reconstruction of the evolutionary history of the mitochondrial proteome across the different eukaryotic lineages. (A) Incoming and outgoing arrows indicate the gains and losses of the MRPs that are indicated in the box. Genes encoding proteins of the bacterial core are shown in bold. The scenario of gene gain and loss was adjusted according to the MRP content encoded by the mitochondrial genomes of rice and R. americana (5) [dashed line, position in tree inferred from (89)]. Dotted lines indicate lineages with highly reduced mitochondria in which all MRP-encoding genes have been lost. Species for which the mitoribosomal proteome has been experimentally investigated are highlighted in a gray shaded box. Numbers following the species names denote the total number of MRPs identified in that species. Note that several MRPs have been lost independently in different lineages. For example, MRPS1 is lost independently in C. reinhardtii, A. thaliana (MRPS1 is encoded on the mitochondrial genomes of Oryza sativa and R. americana) and in the kinetoplastida and alveolata lineages. (B) Evolutionary trajectory towards the human mitochondrial ribosomal proteome. Main gene gain events can be observed at major branching points of the tree, such as in the ancestral eukaryote and at the origin of the metazoa, after which the mitoribosomal proteome is maintained at a constant level. AVE: alveolates, viridiplantae and excavates branch.

Figure 3.

Maximum-likelihood tree of the MRPS18 orthologous group. The metazoa contain three copies of this family (MRPS18A, MRPS18B and MRPS18C) as a result of two consecutive duplication events at the root of the metazoan radiation. Proteins are indicated by a species name and a protein identifier. Bootstrap values for partitions that are supported with values above 50% (out of 100 replicates) are displayed.

Figure 4.

Maximum-likelihood tree showing the origin of supernumerary MRPL48 as a result of a duplication event at the base of the metazoan radiation within the MRPS10 orthologous group. All depicted proteins were connected using PSI-BLAST (see Materials and Methods for details). For instance, a search started with human MRPS10 retrieved both the human MRPL48 (Q96GC5) and the cytosolic SSU ribosomal protein RPS20 (RS20_HUMAN) in the second iteration with E-values of 1e−9 and 0.001, respectively. Proteins are indicated by a species name and a protein identifier. Bootstrap values for partitions that are supported with values above 50% (out of 100 replicates) are displayed.

Figure 5.

Maximum-likelihood tree showing that metazoan-specific supernumerary MRPs MRPS30 and MRPL37 originated as a result of a duplication event at the base of the metazoan radiation. The depicted proteins were connected using PSI-BLAST (see Materials and Methods for details). For instance, a search started with human MRPS30 retrieves human MRPL37 within the first iteration with an E-value of 0.001. All proteins are indicated by a species name and a protein identifier. Bootstrap values for partitions that are supported with values above 50% (out of 100 replicates) are displayed.

Figure 6.

Maximum-likelihood tree indicating common ancestry of supernumerary mitoribosomal protein MRPL45 and Tim44, an essential component of the TIM23/PAM complex, which mediates the translocation of nuclear-encoded proteins across the mitochondrial inner membrane. The tree contains alpha-proteobacterial sequences from which mitochondrial MRPL45 and Tim44 have evolved. Note that there are two paralogous Tim44-like families within the alpha-proteobacteria, resulting from a duplication within that lineage. The depicted proteins were connected using PSI-BLAST as outlined in the Materials and Methods. For instance, a search started with human MRPL45 retrieves the Rhizobium meliloti Type I Tim44-like protein (Q92TE6) in the second iteration with an E-value of 1e−7. The yeast Mba1 and human Tim44 proteins were both retrieved in the third iteration (with E-values of 2e−13 and 2e−10, respectively). Proteins are indicated by a species name and a protein identifier. Bootstrap values for partitions that are supported with values above 50% (out of 100 replicates) are displayed.

Figure 7.

Domain architectures of MRPs to which additional functional domains have been added. Protein identifiers are depicted as in Table 1 or 2 and species distribution is indicated. Proteins are drawn approximately according to scale. Domain abbreviations: (1) Pkinase (pfam00069): Protein kinase catalytic domain. (2) DUF298 (pfam03556): Domain of unknown function, containing a basic helix-loop-helix leucine zipper motif. (3) Collagen (pfam01391): Collagen triple helix repeat. (4) VAR1 (pfam05316): This domain is specific for some fungal mitochondrial ribosomal S3 proteins and is often present in two copies (also see text). (5) AAA (pfam00004): ATPase family associated with various cellular activities. Proteins containing AAA domains often perform chaperone-like functions that assist in the assembly, operation or disassembly of protein complexes. (6) RRM (pfam00076): RNA-binding domain, found in a variety of RNA-binding proteins (also see text). (7) Cu (pfam02298): Copper-binding domain found in various proteins (also see text). (8) In plants, the N-terminus (S3_N, pfam00417) and C-terminus (S3_C, pfam00189) of MRPS3 are separated as a result of an insertion of a protein domain of unknown function (pfam B domain PB012879). (9) COG5373: Predicted membrane protein present in prokaryotes. (10) CBS (pfam00571) and PB1 (pfam00564) domains: CBS domains are small intracellular modules, which have a regulatory role in making proteins sensitive to adenosyl carrying ligands. PB1 is present in many eukaryotic cytoplasmic signaling proteins. (11) COG0021: Transketolase (1-deoxyxylulose-5-phosphate synthase).