Involvement of mitochondrial ribosomal proteins in ribosomal RNA-mediated protein folding

The peptidyl transferase center of the domain V of large ribosomal RNA in the prokaryotic and eukaryotic cytosolic ribosomes acts as general protein folding modulator. We showed earlier that one part of the domain V (RNA1 containing the peptidyl transferase loop) binds unfolded protein and directs it to a folding competent state (FCS) that is released by the other part (RNA2) to attain the folded native state by itself. Here we show that the peptidyl transferase loop of the mitochondrial ribosome releases unfolded proteins in FCS extremely slowly despite its lack of the rRNA segment analogous to RNA2. The release of FCS can be hastened by the equivalent activity of RNA2 or the large subunit proteins of the mitochondrial ribosome. The RNA2 or large subunit proteins probably introduce some allosteric change in the peptidyl transferase loop to enable it to release proteins in FCS.     

YmL9, a nucleus-encoded mitochondrial ribosomal protein of yeast, is homologous to L3 ribosomal proteins from all natural kingdoms and photosynthetic organelles.

The nuclear gene for mitochondrial ribosomal protein YmL9 (MRP-L9) of yeast has been cloned and sequenced. The deduced amino acid sequence characterizes YmL9 as a basic (net charge + 30) protein of 27.5 kDa with a putative signal peptide for mitochondrial import of 19 amino acid residues. The intact MRP-L9 gene is essential for mitochondrial function and is located on chromosome XV or VII. YmL9 shows significant sequence similarities to Escherichia coli ribosomal protein L3 and related proteins from various organisms of all three natural kingdoms as well as photosynthetic organelles (cyanelles). The observed structural conservation is located mostly in the C-terminal half and is independent of the intracellular location of the corresponding genes [Graack, H.-R., Grohmann, L. & Kitakawa, M. (1990) Biol. Chem. Hoppe Seyler 371, 787-788]. YmL9 shows the highest degree of sequence similarity to its eubacterial and cyanelle homologues and is less related to the archaebacterial or eukaryotic cytoplasmic ribosomal proteins. Due to their high sequence similarity to the YmL9 protein two mammalian cytoplasmic ribosomal proteins [MRL3 human and rat; Ou, J.-H., Yen, T. S. B., Wang, Y.-F., Kam, W. K. & Rutter, W. J. (1987) Nucleic Acids Res. 15, 8919-8934] are postulated to be true nucleus-encoded mitochondrial ribosomal proteins.     

Critical function of death-associated protein 3 in T cell receptor-mediated apoptosis induction

Death-associated protein 3 (DAP3) is crucial for promoting apoptosis induced by various stimulations. This report demonstrates that DAP3 is also important for T cell receptor (TCR)-mediated apoptosis induction in immature thymocytes. Enforced expression of DAP3 accelerated the negative selection in developing thymocytes, using the reaggregate thymus organ culture system. In addition, expression of DAP3 accelerated TCR-mediated apoptosis induction in DO11.10 cells. We also demonstrated that DAP3 translocates into the nucleus during TCR-mediated apoptosis in a Nur77 dependent manner. It is concluded that DAP3 is critical for TCR-mediated induction of apoptosis at the downstream of Nur77.     

Identification of phosphorylation sites in mammalian mitochondrial ribosomal protein DAP3

Mammalian mitochondrial ribosomes synthesize 13 proteins that are essential for oxidative phosphorylation. In addition to their role in protein synthesis, some of the mitochondrial ribosomal proteins have acquired functions in other cellular processes such as apoptosis. Death-associated protein 3 (DAP3), also referred to as mitochondrial ribosomal protein S29 (MRP-S29), is a GTP-binding pro-apoptotic protein located in the small subunit of the ribosome. Previous studies have shown that phosphorylation is one of the most likely regulatory mechanisms for DAP3 function in apoptosis and may be in protein synthesis; however, no phosphorylation sites were identified. In this study, we have investigated the phosphorylation status of ribosomal DAP3 and mapped the phosphorylation sites by tandem mass spectrometry. Mitochondrial ribosomal DAP3 is phosphorylated at Ser215 or Thr216, Ser220, Ser251 or Ser252, and Ser280. In addition, phosphorylation of recombinant DAP3 by Protein kinase A and Protein kinase Cdelta at residues that are endogenously phosphorylated in ribosomal DAP3 suggests both of these kinases as potential candidates responsible for the in vivo phosphorylation of DAP3 in mammalian mitochondria. Interestingly, the majority of the phosphorylation sites detected in our study are clustered around the highly conserved GTP-binding motifs, speculating on the significance of these residues on protein conformation and activity. Site-directed mutagenesis studies on selected phosphorylation sites were performed to determine the effect of phosphorylation on cell proliferation and PARP cleavage as indication of caspase activation. Overall, our findings suggest DAP3, a mitochondrial ribosomal small subunit protein, is a novel phosphorylated target.     

A yeast BH3-only protein mediates the mitochondrial pathway of apoptosis

Mitochondrial outer membrane permeabilization is a watershed event in the process of apoptosis, which is tightly regulated by a series of pro- and anti-apoptotic proteins belonging to the BCL-2 family, each characteristically possessing a BCL-2 homology domain 3 (BH3). Here, we identify a yeast protein (Ybh3p) that interacts with BCL-X(L) and harbours a functional BH3 domain. Upon lethal insult, Ybh3p translocates to mitochondria and triggers BH3 domain-dependent apoptosis. Ybh3p induces cell death and disruption of the mitochondrial transmembrane potential via the mitochondrial phosphate carrier Mir1p. Deletion of Mir1p and depletion of its human orthologue (SLC25A3/PHC) abolish stress-induced mitochondrial targeting of Ybh3p in yeast and that of BAX in human cells, respectively. Yeast cells lacking YBH3 display prolonged chronological and replicative lifespans and resistance to apoptosis induction. Thus, the yeast genome encodes a functional BH3 domain that induces cell death through phylogenetically conserved mechanisms.     

Another function for the mitochondrial ribosomal RNA: protein folding

Specialized proteins known as molecular chaperones bind transiently to non-native conformational states of proteins and protein complexes to promote transition to a biologically active conformation. Recently, it was demonstrated in vitro that proteins do not uniquely possess this activity. We show that mitochondrial 12S and 16S ribosomal RNA can fold chemically denatured proteins and reactivate heat-induced aggregated proteins in vitro. This chaperone action is ATP-independent. The specific secondary structure of the mitochondrial rRNA is critical to its folding activity. Furthermore, mutant mitochondrial 16S rRNA from aged cardiac muscle cells lacked this activity. We propose that mitochondrial 12S and 16S ribosomal RNA may play an important role in protein folding in mitochondria.     

Contacts between mammalian mitochondrial translational initiation factor 3 and ribosomal proteins in the small subunit

Mammalian mitochondrial translational initiation factor 3 (IF3(mt)) binds to the small subunit of the ribosome displacing the large subunit during the initiation of protein biosynthesis. About half of the proteins in mitochondrial ribosomes have homologs in bacteria while the remainder are unique to the mitochondrion. To obtain information on the ribosomal proteins located near the IF3(mt) binding site, cross-linking studies were carried out followed by identification of the cross-linked proteins by mass spectrometry. IF3(mt) cross-links to mammalian mitochondrial homologs of the bacterial ribosomal proteins S5, S9, S10, and S18-2 and to unique mitochondrial ribosomal proteins MRPS29, MRPS32, MRPS36 and PTCD3 (Pet309) which has now been identified as a small subunit ribosomal protein. IF3(mt) has extensions on both the N- and C-termini compared to the bacterial factors. Cross-linking of a truncated derivative lacking these extensions gives the same hits as the full length IF3(mt) except that no cross-links were observed to MRPS36. IF3 consists of two domains separated by a flexible linker. Cross-linking of the isolated N- and C-domains was observed to a range of ribosomal proteins particularly with the C-domain carrying the linker which showed significant cross-linking to several ribosomal proteins not found in prokaryotes.     

Proteomics of bovine mitochondrial RNA-binding proteins: HES1/KNP-I is a new mitochondrial resident protein

Proteomic analysis of bovine mitochondrial proteins with affinity to polyAdenylate or polyUridylate was performed in an effort to identify novel RNA-binding mitochondrial proteins. We have used 2D gel electrophoresis and MALDI-QqTOF mass spectrometry to identify a total of 64 proteins, of which 51 have defined mitochondrial function including 6 known RNA-binding proteins. HES1/KNP-I from the polyA-binding fraction of mitochondrial Triton extract showed exclusive mitochondrial localization when expressed in GFP-tagged form. The HES1/KNP-I gene is on human chromosome 21q22.3 and may be involved in several disorders mapped to that region. Thus, HES1/KNP-I is a proven mitochondrial resident protein with apparent tight membrane association and tentative RNA-binding properties.     

Polypyrimidine tract-binding proteins are cleaved by caspase-3 during apoptosis

The polypyrimidine tract-binding protein (PTB), an RNA-binding protein, is required for efficient translation of some mRNAs containing internal ribosomal entry sites (IRESs). Here we provide evidence that the addition of apoptosis-inducing agents to cells results in the cleavage of PTB isoforms 1, 2, and 4 by caspase-3. This cleavage of PTB separated the N-terminal region, containing NLS-RRM1, from the C-terminal region, containing RRM2-3-4. Our data indicate that there are three noncanonical caspase-3 target sites in PTBs, namely Ile-Val-Pro-Asp(7)Ile, Leu-Tyr-Thr-Asp(139)Ser, and Ala-Ala-Val-Asp(172)Ala. The C-terminal PTB fragments localized to the cytoplasm, as opposed to the nucleus where most intact PTBs are found. Moreover, these C-terminal PTB fragments inhibited translation of polioviral mRNA, which contains an IRES element requiring PTB for its activation. This suggests that translation of some IRES-containing mRNAs is regulated by proteolytic cleavage of PTB during apoptosis.     

Identification of 12 new yeast mitochondrial ribosomal proteins including 6 that have no prokaryotic homologues

Mitochondrial ribosomal proteins were studied best in yeast, where the small subunit was shown to contain about 35 proteins. Yet, genetic and biochemical studies identified only 14 proteins, half of which were predictable by sequence homology with prokaryotic ribosomal components of the small subunit. Using a recently described affinity purification technique and tagged versions of yeast Ykl155c and Mrp1, we isolated this mitochondrial ribosomal subunit and identified a total of 20 proteins, of which 12 are new. For a subset of the newly described ribosomal proteins, we showed that they are localized in mitochondria and are required for the respiratory competency of the yeast cells. This brings to 26 the total number of proteins described as components of the mitochondrial small ribosomal subunit. Remarkably, almost half of the previously and newly identified mitochondrial ribosomal components showed no similarity to any known ribosomal protein. Homologues could be found, however, in predicted protein sequences from Schizosaccharomyces pombe. In more distant species, putative homologues were detected for Ykl155c, which shares conserved motifs with uncharacterized proteins of higher eukaryotes including humans. Another newly identified ribosomal protein, Ygl129c, was previously shown to be a member of the DAP-3 family of mitochondrial apoptosis mediators.     

Genes for two mitochondrial ribosomal proteins in flowering plants are derived from their chloroplast or cytosolic counterparts

Often during flowering plant evolution, ribosomal protein genes have been lost from the mitochondrion and transferred to the nucleus. Here, we show that substitution by a duplicated, divergent gene originally encoding the chloroplast or cytosolic ribosomal protein counterpart accounts for two missing mitochondrial genes in diverse angiosperms. The rps13 gene is missing from the mitochondrial genome of many rosids, and a transferred copy of this gene is not evident in the nucleus of Arabidopsis, soybean, or cotton. Instead, these rosids contain a divergent nuclear copy of an rps13 gene of chloroplast origin. The product of this gene from all three rosids was shown to be imported into isolated mitochondria but not into chloroplasts. The rps8 gene is missing from the mitochondrion and nucleus of all angiosperms examined. A divergent copy of the gene encoding its cytosolic counterpart (rps15A) was identified in the nucleus of four angiosperms and one gymnosperm. The product of this gene from Arabidopsis and tomato was imported successfully into mitochondria. We infer that rps13 was lost from the mitochondrial genome and substituted by a duplicated nuclear gene of chloroplast origin early in rosid evolution, whereas rps8 loss and substitution by a gene of nuclear/cytosolic origin occurred much earlier, in a common ancestor of angiosperms and gymnosperms.     

Bovine TWINKLE and mitochondrial ribosomal protein L43 genes are regulated by an evolutionary conserved bidirectional promoter

TWINKLE is a mitochondrial DNA helicase playing an important role in mitochondrial DNA replication. In human, mutations in this gene cause progressive external ophtalmoplegia and mitochondrial DNA depletion syndrome-7. TWINKLE is well conserved among multicellular eukaryotes and is believed to be a key regulator of mitochondrial DNA copy number in mammals.     

A proteomics approach to the identification of mammalian mitochondrial small subunit ribosomal proteins

Mammalian mitochondrial small subunit ribosomal proteins were separated by two-dimensional polyacrylamide gel electrophoresis. The proteins in six individual spots were subjected to in-gel tryptic digestion. Peptides were separated by capillary liquid chromatography, and the sequences of selected peptides were obtained by electrospray tandem mass spectrometry. The peptide sequences obtained were used to screen human expressed sequence tag data bases, and complete consensus cDNAs were assembled. Mammalian mitochondrial small subunit ribosomal proteins from six different classes of ribosomal proteins were identified. Only two of these proteins have significant sequence similarities to ribosomal proteins from prokaryotes. These proteins correspond to Escherichia coli S10 and S14. Homologs of two human mitochondrial proteins not found in prokaryotes were observed in the genomes of Drosophila melanogaster and Caenorhabditis elegans. A homolog of one of these proteins was observed in D. melanogaster but not in C. elegans, while a homolog of the other was present in C. elegans but not in D. melanogaster. A homolog of one of the ribosomal proteins not found in prokaryotes was tentatively identified in the yeast genome. This latter protein is the first reported example of a ribosomal protein that is shared by mitochondrial ribosomes from lower and higher eukaryotes that does not have a homolog in prokaryotes.     

Uncoupling proteins 2 and 3 are fundamental for mitochondrial Ca2+ uniport

Mitochondrial Ca(2+) uptake is crucial for the regulation of the rate of oxidative phosphorylation, the modulation of spatio-temporal cytosolic Ca(2+) signals and apoptosis. Although the phenomenon of mitochondrial Ca(2+) sequestration, its characteristics and physiological consequences have been convincingly reported, the actual protein(s) involved in this process are unknown. Here, we show that the uncoupling proteins 2 and 3 (UCP2 and UCP3) are essential for mitochondrial Ca(2+) uptake. Using overexpression, knockdown (small interfering RNA) and mutagenesis experiments, we demonstrate that UCP2 and UCP3 are elementary for mitochondrial Ca(2+) sequestration in response to cell stimulation under physiological conditions - observations supported by isolated liver mitochondria of Ucp2(-/-) mice lacking ruthenium red-sensitive Ca(2+) uptake. Our results reveal a novel molecular function for UCP2 and UCP3, and may provide the molecular mechanism for their reported effects. Moreover, the identification of proteins fundemental for mitochondrial Ca(2+) uptake expands our knowledge of the physiological role for mitochondrial Ca(2+) sequestration.