Mammalian Mitochondrial Protein Research Articles

Mammalian Mitochondrial Ribosomal Proteins (4): AMINO ACID SEQUENCING, CHARACTERIZATION, AND IDENTIFICATION OF CORRESPONDING GENE SEQUENCES

Mitochondrial ribosomal proteins (MRPs) are required for the translation of all 13 mitochondrial encoded genes in humans. It has been speculated that mutations and polymorphisms in the human MRPs may be a primary cause of some oxidative phosphorylation disorders or modulate the severity and tissue specificity of pathogenic mitochondrial DNA mutations. Although the sequences of most of the yeast MRPs are known, only very few mammalian and nearly no human MRPs have been completely characterized. MRPs differ greatly in sequence, and sometimes biochemical properties, between different species, not allowing easy recognition by sequence homology. Therefore, the Mammalian Mitochondrial Ribosomal Consortium is using a direct approach of purifying individual mammalian (bovine) MRPs, determining their N-terminal and/or internal peptide sequences using different protein sequencing techniques, and using the resulting sequence information for screening expressed sequence tags and genomic data bases to determine human, mouse, and rat homologues of the bovine proteins. Two proteins of the large and three proteins of the small ribosomal subunit have been analyzed in this manner. Three of them represent "new," i.e. formerly unknown mammalian mitochondrial ribosomal protein classes. Only one of these three different MRPs shows significant sequence similarities to known ribosomal proteins. In one case, the corresponding human genomic DNA sequences were found in the data bases, and the exon/intron structure was determined.

Mammalian Mitochondrial Ribosomal Proteins (2): AMINO ACID SEQUENCING, CHARACTERIZATION, AND IDENTIFICATION OF CORRESPONDING GENE SEQUENCES

Four different classes of mammalian mitochondrial ribosomal proteins were identified and characterized. Mature proteins were purified from bovine liver and subjected to N-terminal or matrix-assisted laser-desorption mass spectroscopic amino acid sequencing after tryptic in-gel digestion and high pressure liquid chromatography separation of the resulting peptides. Peptide sequences obtained were used to virtually screen expressed sequence tag data bases from human, mouse, and rat. Consensus cDNAs were assembled in silico from various expressed sequence tag sequences identified. Deduced mammalian protein sequences were characterized and compared with ribosomal protein sequences of Escherichia coli and yeast mitochondria. Significant sequence similarities to ribosomal proteins of other sources were detected for three out of four different mammalian protein classes determined. However, the sequence conservation between mitochondrial ribosomal proteins of mammalian and yeast origin is much less than the sequence conservation between cytoplasmic ribosomal proteins of the same species. In particular, this is shown for the mammalian counterparts of the E. coli EcoL2 ribosomal protein (MRP-L14), that do not conserve the specific and functional highly important His(229) residue of E. coli and the corresponding yeast mitochondrial Rml2p.

Mammalian mitochondrial extracts possess DNA end-binding activity.

Mammalian mitochondrial protein extracts possess DNA end-binding (DEB) activity. Protein binding to a 394 bp double-stranded DNA molecule was measured using an electrophoretic mobility shift assay. Mitochondrial DEB activity was highly specific for linear DNA. Inclusion of a vast excess of non-radioactive circular DNA did not disrupt binding to radioactive f394. In contrast, binding was abolished by the inclusion of linear competitor DNA. In mammals, nuclear DEB activity is due to Ku, a hetero-dimer composed of the Ku70 and Ku86 proteins. To determine whether mitochondrial DEB activity was also due to Ku, protein extracts were prepared from the Chinese hamster XR-V15B cell line, which lacks this protein. As anticipated, nuclear extracts prepared from these cells lacked DEB activity. In contrast, mitochondrial extracts prepared from these cells had wild-type levels of DEB activity, demonstrating that this latter activity is not a consequence of nuclear contamination. Although the nuclear and mitochondrial DEB activities are independent of each other, they are nevertheless closely related, since mitochondrial DEB activity was 'supershifted' by both anti-Ku70 and anti-Ku86 antisera. The nuclear DEB protein Ku plays an essential role in nuclear DNA double-strand break repair. The DEB activity described herein may therefore play a similar role in mitochondrial DNA repair.

Identification and characterization of a mammalian mitochondrial ATP-binding cassette membrane protein

Membrane proteins of the ATP-binding cassette (ABC) superfamily are involved in the transport of diverse substrates across organellar and plasma membranes of the mammalian cell. Most human ABC proteins identified to date are associated with genetically linked diseases or clinically relevant phenotypes. We describe a new human half-molecule ABC protein, designated M-ABC1, that contains a predicted single membrane and ATP-binding cassette domain. M-ABC1 is localized to membranes of the mitochondria and its transcript is expressed in all tissues. The N-terminal region of the M-ABC1 protein was shown to function independently as a mitochondrial signal sequence by its ability to target the green fluorescent protein to the mitochondria. The monomeric 60 kDa M-ABC1 protein was chemically crosslinked in vivo into a major protein species of 120-130 kDa, thereby confirming that M-ABC1 exists within a higher ordered ABC protein complex. A dominant negative repression approach using M-ABC1 protein with site-directed mutations in its Walker A motif revealed that the mutant protein was rapidly degraded and indicated that the intact Walker A motif of M-ABC1 was required for its stability. The identification of M-ABC1 extends the known distribution of members of the ABC protein family into the mammalian mitochondrion.

Mammalian Mitochondrial Ribosomal Proteins: N-TERMINAL AMINO ACID SEQUENCING, CHARACTERIZATION, AND IDENTIFICATION OF CORRESPONDING GENE SEQUENCES

The integrity of healthy mitochondria is supposed to depend largely on proper mitochondrial protein biosynthesis. Mitochondrial ribosomal proteins (MRPs) are directly involved in this process. To identify mammalian mitochondrial ribosomal proteins and their corresponding genes, we purified mature rat MRPs and determined 12 different N-terminal amino acid sequences. Using this peptide information, data banks were screened for corresponding DNA sequences to identify the genes or to establish consensus cDNAs and to characterize the deduced MRP open reading frames. Eight different groups of corresponding mammalian MRPs constituted from human, mouse, and rat origin were identified. Five of them show significant sequence similarities to bacterial and/or yeast mitochondrial ribosomal proteins. However, MRPs are much less conserved in respect to the amino acid sequence among species than cytoplasmic ribosomal proteins of eukaryotes and bacteria.

Evidence for the Existence of Distinct Mammalian Cytosolic, Microsomal, and Two Mitochondrial GrpE-like Proteins, the Co-chaperones of Specific Hsp70 Members

We previously reported the cDNA cloning and characterization of a mammalian mitochondrial GrpE protein ( approximately 21 kDa, mt-GrpE#1) and now provide evidence for the presence of distinct cytosolic ( approximately 40 kDa), microsomal ( approximately 50 kDa), and additional mitochondrial ( approximately 22 kDa, mt-GrpE#2) GrpE-like members. While a cytosolic GrpE-like protein has recently been identified, the demonstration of both a microsomal and a second mitochondrial GrpE-like member represents the first in any biological system. Investigation of the microsomal and two mitochondrial GrpE-like proteins revealed that they bound specifically to Escherichia coli DnaK, and the complexes formed were not disrupted in the presence of 0.5 M salt but were readily dissociated in the presence of 5 mM ATP. The functional integrity of mt-GrpE#1 and #2 was verified by their ability to specifically interact with and stimulate the ATPase activity of mammalian mitochondrial Hsp70 (mt-Hsp70). Analysis of the cDNA sequences encoding the two mammalian mitochondrial GrpE-like proteins revealed approximately 47% positional identity at the amino acid level, the presence of a highly conserved mitochondrial leader sequence, and putative destabilization elements within the 3'-untranslated region of the mt-GrpE#2 transcript which are not present in the mt-GrpE#1 transcript. A constitutive expression of both mitochondrial GrpE-like transcripts in 22 distinct mouse tissues was observed but possible different post-transcriptional regulation of the mt-GrpE#1 and #2 transcripts may confer a different expression pattern of the encoded proteins.

A Delayed-early Response Nuclear Gene Encoding MRPL12, the Mitochondrial Homologue to the Bacterial Translational Regulator L7/L12 Protein

We have characterized a new delayed-early response mRNA encoding a 21-kDa product (MRPL12) that accumulates during the G1 phase of growth-stimulated cells. MRPL12 is the mammalian homologue to chloroplastic and bacterial L12 ribosomal proteins. Immunofluorescence microscopy and cell fractionation indicate a predominant mitochondrial localization in various mammalian cell lines. The NH2-terminal 49 amino acids are necessary and sufficient to target the protein within the mitochondria and are probably cleaved off during import. MRPL12 proteins associated in vitro and cofractionate with ribosomal structures, as is the case for prokaryotic L12 proteins. Expression of a dominant inhibitory truncated protein leads to a severe reduction in cell growth by inhibiting mitochondrial ATP production. MRPL12 is the first mammalian mitochondrial ribosomal protein to be characterized.

Cloning, sequence analysis and expression of mammalian mitochondrial protein synthesis elongation factor Tu

The bovine liver mitochondrial protein synthesis elongation factor Tu · Ts complex (EF-Tu · Ts mt) has been purified and partial peptide sequence information has been obtained for EF-TU mt. A complete cDNA has been obtained encoding bovine EF-Tu mt and a nearly complete cDNA has been obtained for human EF-Tum mt. The bovine cDNA has a 5′ untranslated leader, an open reading frame of 1356 nucleotides and a 3′ untranslated region of 189 base pairs. NH 2-terminal sequencing of the mature protein indicates that the transit peptide for the mitochondrial localization of this protein is 43 amino acids in length. The human and bovine factors are 95% identical. The deduced protein sequences show considerable identity to bacterial and organellar EF-Tu sequences. At least two genes for EF-Tu mt are present in the bovine system. Northern analysis indicates that EF-Tu mt is synthesized in all tissues but that the level of expression varies over a wide range. EF-Tu mt has been expressed in E. coli as a His-tagged protein and purified to near homogeneity. The expressed form of the factor is active in the poly(U)-directed polymerization of phenylalanine although it is less active than the native EF-Tu · Ts mt complex.

The two mammalian mitochondrial stress proteins, grp 75 and hsp 58, transiently interact with newly synthesized mitochondrial proteins.

In mammalian cells, two of the so-called heat shock (hsp) or stress proteins are components of the mitochondria. One of these, hsp 58, is a member of the bacterial GroEL family, whereas the other, glucose-regulated protein (grp) 75, represents a member of the hsp 70 family of stress proteins. Owing to previous studies implicating a role for both the hsp 70 and GroEL families in facilitating protein maturation events, we used the method of native immunoprecipitation to examine whether hsp 58 and grp 75 might interact with other proteins of the mitochondria. In cells pulse-labeled with [35S]-methionine, a significant number of newly synthesized mitochondrial proteins co-precipitated with either hsp 58 or grp 75. Such interactions appeared transient. For example, providing the pulse-labeled cells a subsequent chase period in the absence of radiolabel resulted in a reduction of co-precipitating proteins. If the pulse-chase labeling experiments were performed in the presence of an amino acid analogue, somewhat different results were obtained. Specifically, although many of the newly synthesized and analogue-containing proteins again were observed to co-precipitate with grp 75, the interactions did not appear transient, but instead were stable. Under steady-state labeling conditions, we also observed a portion of hsp 58 and grp 75 in an apparent complex with one another. On addition of ATP, the complex was dissociated. Accompanying this dissociation was the concomitant autophosphorylation of grp 75. On the basis of these observations, as well as previous studies examining the structure/function of the hsp 70 and GroEL proteins, we suspect that both hsp 58 and grp 75 interact with and facilitate the folding and assembly of proteins as they enter into the mitochondria.

Experimental approaches to the study of the biogenesis of mammalian mitochondrial proteins.

Mitochondrial proteins are synthesized in mitochondria and on cytosolic ribosomes. Several approaches used to establish the site of synthesis and the identity of mitochondrially synthesized proteins are described. These include the specific inhibition of mitochondrial translation by inhibitors or mutation and the specific elimination of cytosolic translation either by using isolated mitochondria or specific inhibitors. Experimental approaches to study the import of proteins into mitochondria are also discussed.

Chloramphenicol

Chloramphenicol was introduced into medical practice in 1949. At therapeutic concentrations of 10 to 20 micrograms drug per ml, the drug inhibits bacterial ribosomal and, to a lesser extent, mammalian mitochondrial protein synthesis but concentrations above 60 micrograms drug per ml induce progressive reduction of oxygen-dependent cellular metabolism. Some adverse reactions (e.g. bone marrow suppression and the "gray baby syndrome") reflect these effects. The pathogenesis of chloramphenicol-induced aplastic anemia remains unclear. Chloramphenicol is most bioavailable after oral administration and has a remarkable ability to diffuse into body fluids and tissues. However, there are wide interindividual variations in its metabolism and elimination, particularly in newborns. Chloramphenicol is indicated for invasive ampicillin-resistant H. influenzae infections; for patients allergic to penicillin with pneumococcal, meningococcal or H. influenzae meningitis; in patients under 8 years of age with Rocky Mountain spotted fever; and for the treatment of brain abscess and other severe anaerobic infections (excluding endocarditis) due to B. fragilis. Other indications include selected patients with Salmonella meningitis or carditis, rickettsioses and intraocular infections. Unravelling the pathogenesis of chloramphenicol-induced aplastic anemia is critical to more widespread application of this remarkable antimicrobial.

Control of yeast and mammalian mitochondrial protein synthesis by cytoplasmic factors

The biogenesis of the mitochondrial inner membrane requires the synthesis of proteins at 2 intracellular sites: 1 cytoplasmic; 1 mitochondrial. A mechanism must exist to coordinate the synthesis of proteins at the 2 distinct intracellular sites such that mitochondrial formation occurs in an orderly manner. Previous studies in yeast using selective inhibitors of cytoplasmic and mitochondrial protein synthesis have suggested that cytoplasmically synthesized proteins may control mitochondrial protein synthesis both in vivo [l-4] and in vitro [3] and that this control may occur by either stimulation of chain initiation or increased formation of mitochondrial mRNAs [5]. In [6] protein synthesis by yeast mitochondria in vitro was reported stimulated by addition of a high-speed cytoplasmic supernatant from yeast [6]. This effect is not species-specific, as protein synthesis in vitro by isolated yeast mitochondria could be stimulated markedly by post-polysomal supernatants from either E. coli, rat liver or yeast [7]. However, stimulation of yeast mitochondrial protein synthesis by all of these high speed supernatants may be due to the presence of non-dialyzable GMP which is converted to GDP (or GTP) during the incubation in the protein-synthesizing medium [8]. We have confirmed that GTP stimulates yeast mitochondrial protein synthesis in vitro, but addition of a post-polysomal supernatant from yeast doubles the rate of mitochondrial protein synthesis in

Differences in the mitochondrially synthesized subunits of human and mouse cytochrome c oxidase

Ten or so proteins synthesized in mammalian mitochondria in vivo can be resolved by electrophoresis in polyacrylamide gels, after solubilization with sodium dodecyl sulphate [ I] . Using interspecific differences in the electrophoretic pattern of these proteins, we analysed the nature of the mitochondrially made proteins which are synthesized in human-mouse somatic cell hybrids. The observed patterns of these proteins in such cells suggested that interspecific variations in mitochondrially synthesized proteins are determined extrachromosomally and therefore possibly by mitochondrial DNA [2] . We have attempted to discover the functions of some of these products of mammalian mitochondrial protein synthesis, and now report that one of them, which shows considerable interspecific variation, appears to be associated with the cytochrome c oxidase (EC 1.9.3.1) complex of mammalian mitochondria.

Initiation of mammalian mitochondrial protein synthesis the effect of methotrexate

Protein synthesis in mammalian cell mitochondria is initiated with fMet-tRNA Met f, the formyl group being most likely donated by N 10-formyltetrahy-drofolate. Methotrexate, which depletes the C 1-folate pools by inhibiting dihydrofolate reductase, might block the formylation reaction, inhibiting mitochondrial protein synthesis and indirectly cell growth. L cells grown in suspension culture are found to grow normally in the presence of 5 · 10 −5 M methotrexate when supplied with metabolites whose synthesis requires the C 1-folate pools. Preincubation for 15 min with 10 −4 M methotrexate does not affect the subsequent formylation of mitochondrial initiator tRNA. Also, there is no effect on the synthesis of N- formylmethionylpuromycin in L cells grown for 160 h in the presence of 5 · 10 −6 M methotrexate, nor in KB cells grown for 72 h in its presence. These results demonstrate that cell growth, the formylation of mitochondrial initiator tRNA and the initiation of mitochondrial protein synthesis are not inhibited by methotrexate when cells are supplied with metabolites whose de novo synthesis requires C 1 -folate pools. The mechanism of continued formylation in the presence of methotrexate is not known.

The interference of the macrolide antibiotics with mitochondrial protein synthesis

1. Protein synthesis by isolated BHK-21 mitochondria was shown to be inhibited by the macrolides erythromycin, tylosin tartrate, spiramycin and carbomycin. Intact mitochondria from rat liver were insensitive to erythromycin, but inhibited by tylosin tartrate, spiramycin and carbomycin. 2. Cytochrome c oxidase formation in BHK-21 cells was unaffected by tylosin tartrate and spiramycin, but strongly inhibited by carbomycin. Mitochondrial protein synthesis in whole BHK-21 cells showed the same insensitivity to tylosin tartrate and spiramycin. 3. Peptidyl transferase activity of isolated rat-liver mitochondrial ribosomes was inhibited by tylosin tartrate, spiramycin and carbomycin, whereas chloramphenicol inhibition was reversed by erythromycin. Rather high antibiotic concentrations (200 μg/ml or higher) were necessary to obtain these effects. For Neurospora crassa mitochondrial ribosomes a similar response to these macrolides was found. Therefore mitochondrial ribosomes from N. crassa are markedly less sensitive to macrolides than yeast mitochondrial ribosomes. 4. It is shown that mammalian mitochondrial protein synthesis is sensitive to erythromycin, tylosin tartrate, spiramycin and carbomycin, but that for the first three antibiotics membrane barriers exist: for erythromycin the mitochondrial membrane is the barrier and for tylosin tartrate and spiramycin the cell membrane. 5. Amino acid incorporation by isolated mitochondria appears much more sensitive to inhibition by tylosin tartrate, spiramycin and carbomycin than the isolated 55-S ribosomes. For carbomycin this can be explained in part by the finding that carbomycin was concentrated by intact rat-liver mitochondria in vitro.

Phylogenetic differences in the sensitivity of mitochondrial protein synthesising systems to antibiotics

The inhibitory action of chloramphenicol on the incorporation of amino acids into protein by isolated mammalian mitochondria (rat liver, beef heart) has been clearly demonstrated in a number of laboratories [l-4] . More recently we have shown that the inhibitory action of chloramphenicol on the mitochondrial protein synthesising system of mammalian tissues also occurs in viva, and results in the selective inhibition of the synthesis of the mitochondrial cytochromes a, a3, t, and cl in HeLa cells and regenerating rat liver [5,6]. It has also been established that the mitochondrial protein synthesising system of yeast is sensitive in viuo and in vitro not only to chloramphenicol but also to lincomycin and the macrolide antibiotics such as erythromycin [7,8], so that its sensitivity to antibiotics resembles that of the bacterial ribosome, a finding which suggested that mitochondrial protein synthesising systems in general may be sensitive to this wide range of antibiotics. However, this communication reports that significant differences exist in the antibiotic sensitivity of the mitochondrial protein synthesising systems of yeast and mammalian mitochondria. Although chloramphenicol inhibits amino acid incorporation into protein by mitochondria from the mammalian species investigated (rat, rabbit and cat), the incorporation is insensitive to both erythromycin and lincomycin. The results are interpreted to suggest that in the course of the evolution of the mammalian mitochondrial protein synthesising system an intrinsic change in the system has occurred which reflects a change in cytoplasmic genetic information.