Characterization of a mutation responsible for an alkali-sensitive mutant, 18224, of alkaliphilic Bacillus sp. strain C-125

An alkali-sensitive mutant, 18224, of the alkaliphilic Bacillus sp. strain C-125 was characterized. The nucleotide sequence of the PvuI-NlaIV DNA fragment that recovers the alkaliphily of 18224 has been cloned from the mutant and sequenced. Comparison of the nucleotide sequences of the corresponding regions found a G to A substitution in the mutant. The mutation resulted in an amino acid substitution from 82Gly to Glu of the putative ORF3 product, which consisted a gene cluster of at least four tandemly located open reading frames. The ORF3 product was deduced to be an 112 amino acid polypeptide with hydrophobic properties, which was expressed using an in vitro translation system.     

Import into mitochondria, folding and retrograde movement of fumarase in yeast

A single translation product of the FUM1 gene encoding fumarase is distributed between the cytosol and mitochondria of Saccharomyces cerevisiae. All fumarase translation products are targeted and processed in mitochondria before distribution. Here we show that targeting of fumarase is coupled to translation and initially involves insertion of the protein across the mitochondrial membranes and processing by the matrix protease. Rapid folding of fumarase may determine its requirement for coupling of its translocation with translation and unique route of distribution. The amino termini of most fumarase molecules are translocated across the mitochondrial membranes and processed. Unlike the in vivo situation where these molecules are released into the cytosol, in vitro they remain externally attached to the mitochondria, thereby positioned for release from the organelle. Our model suggests that fumarase displays a unique mechanism of targeting and distribution, which occurs cotranslationally and involves folding and retrograde movement of the processed protein back through the translocation pore.     

Regulation of Schwann cell proliferation in cultured segments of the adult rat sciatic nerve

A gene coding for a protein that shows homologies to prokaryotic ribosomal protein S2 is present in the mitochondrial (mt) genome of wheat (Triticum aestivum). The wheat gene is transcribed as a single mRNA which is edited by C-to-U conversions at seven positions, all resulting in alteration of the encoded amino acid. Homologous gene sequences are also present in the mt genomes of rice and maize, but we failed to identify the corresponding sequences in the mtDNA of all dicotyledonous species tested; in these species the mitochondrial RPS2 is probably encoded in the nucleus. The protein sequence deduced from the wheat rps2 gene sequence has a long C-terminal extension when compared to other prokaryotic RPS2 sequences. This extension presents no similarity with any known sequence and is not conserved in the maize or rice mitochondrial rps2 gene. Most probably, after translation, this peptide extension is processed by a specific peptidase to give rise to the mature wheat mitochondrial RPS2.     

Magnetic resonance spectroscopy for assessing myocardial rejection in the transplanted rat heart

Tandem duplication of a mitochondrial import leader sequence has been shown to markedly increase the efficiency of translocation of chimaeric precursors across mitochondrial membranes to the mitochondrial matrix. The principle of leader sequence duplication was applied to the protein secretion system of the yeast Saccharomyces cerevisiae and of Bacillus subtilis. The secretion signal sequences of yeast invertase and B. subtilis neutral protease were used to direct the secretion of human interferon alpha 4. Our results show that the duplication of these N-terminal signal sequences does not enhance secretion of interferon alpha 4 in either of the cell systems studied.     

Regulation of mitochondrial iron accumulation by Yfh1p, a putative homolog of frataxin

The gene responsible for Friedreich's ataxia, a disease characterized by neurodegeneration and cardiomyopathy, has recently been cloned and its product designated frataxin. A gene in Saccharomyces cerevisiae was characterized whose predicted protein product has high sequence similarity to the human frataxin protein. The yeast gene (yeast frataxin homolog, YFH1) encodes a mitochondrial protein involved in iron homeostasis and respiratory function. Human frataxin also was shown to be a mitochondrial protein. Characterizing the mechanism by which YFH1 regulates iron homeostasis in yeast may help to define the pathologic process leading to cell damage in Friedreich's ataxia.     

Differential effects of chronic ethanol consumption on hepatic mitochondrial and cytoplasmic ribosomes

The effects of chronic ethanol consumption on the properties of mitochondrial and cytoplasmic ribosomes were investigated in rat liver. Sedimentation properties of purified mitochondrial (55S) and cytoplasmic (80S) ribosomes were determined by analyses on sucrose density gradients. Mitochondrial ribosomes from control animals moved further in the gradients than did those isolated from ethanol-fed rats, which suggests that ethanol ribosomes have a lower molecular weight. In addition, mitochondrial from ethanol-fed animals contained a lower percentage of ribosomes present as the intact monosome, suggesting that ethanol may have an effect on the stability of the functional mitochondrial ribosomes. This was confirmed by the presence of the larger 39S subunit in preparations from ethanol-fed animals. No such ethanol-related alterations were seen with cytoplasmic ribosomes. The protein composition of mitochondrial cytoplasmic ribosomes was investigated using two-dimensional gel electrophoresis, followed by two-dimensional densitometry. As indicated by differences in protein staining intensity, ethanol consumption seemed to alter the concentration of seven mitochondrial ribosomal proteins. In contrast, no such changes were observed in the protein pattern from cytoplasmic ribosomes. Observations in this study provide for the possibility that alterations in the amounts of selected proteins in the mitochondrial ribosome lead to impaired assembly of the ribosome. These ethanol-related structural changes may be responsible for the decreased activity of mitochondrial ribosomes that results in impaired hepatic mitochondrial protein synthesis (W.B. Coleman and C.C. Cunningham, Biochim. Biophys, Acta 1058:178-186, 1991). Furthermore, this study reemphasizes the increased susceptibility of the hepatic mitochondrial translation system, compared with the cytoplasmic system to chronic ethanol consumption.     

Different mechanisms for suppression of apoptosis by cytokines and calcium mobilizing compounds

We addressed the question of whether both mitochondrial and cytoplasmic translation activities decreased simultaneously in human skin fibroblasts with the age of the donors and found that the age-related reduction was limited to mitochondrial translation. Then, to determine which genome, mitochondrial or nuclear, was responsible for this age-related, mitochondria-specific reduction, pure nuclear transfer was carried out from mitochondrial DNA (mtDNA)-less HeLa cells to four fibroblast lines, two from aged subjects, one from a fetus, and one from a patient with cardiomyopathy, and their nuclear hybrid clones were isolated. A normal fibroblast line from the fetus and a respiration-deficient fibroblast line from the patient were used as a positive and a negative control, respectively. Subsequently, the mitochondrial translation and respiration properties of the nuclear hybrid clones were compared. A negative control experiment showed that this procedure could be used to isolate even nuclear hybrids expressing overall mitochondrial respiration deficiency, whereas no respiration deficiencies were observed in any nuclear hybrids irrespective of whether their mtDNAs were exclusively derived from aged or fetal donors. These observations suggest that nuclear-recessive mutations of factors involved in mitochondrial translation but not mtDNA mutations are responsible for age-related respiration deficiency of human fibroblasts.     

Isolation of a cDNA encoding human holocarboxylase synthetase by functional complementation of a biotin auxotroph of Escherichia coli

Holocarboxylase synthetase (HCS) catalyzes the biotinylation of the four biotin-dependent carboxylases in human cells. Patients with HCS deficiency lack activity of all four carboxylases, indicating that a single HCS is targeted to the mitochondria and cytoplasm. We isolated 21 human HCS cDNA clones, in four size classes of 2.0-4.0 kb, by complementation of an Escherichia coli birA mutant defective in biotin ligase. Expression of the cDNA clones promoted biotinylation of the bacterial biotinyl carboxyl carrier protein as well as a carboxyl-terminal fragment of the alpha subunit of human propionyl-CoA carboxylase expressed from a plasmid. The open reading frame encodes a predicted protein of 726 aa and M(r) 80,759. Northern blot analysis revealed the presence of a 5.8-kb major species and 4.0-, 4.5-, and 8.5-kb minor species of poly(A)+ RNA in human tissues. Human HCS shows specific regions of homology with the BirA protein of E. coli and the presumptive biotin ligase of Paracoccus denitrificans. Several forms of HCS mRNA are generated by alternative splicing, and as a result, two mRNA molecules bear different putative translation initiation sites. A sequence upstream of the first translation initiation site encodes a peptide structurally similar to mitochondrial presequences, but it lacks an in-frame ATG codon to direct its translation. We anticipate that alternative splicing most likely mediates the mitochondrial versus cytoplasmic expression, although the elements required for directing the enzyme to the mitochondria remain to be confirmed.     

MBA1 encodes a mitochondrial membrane-associated protein required for biogenesis of the respiratory chain

The yeast MBA 1 gene (Multi-copy Bypass of AFG3) is one of three genes whose overexpression suppresses afg3-null and rca1-null mutations. Bypass of AFG3 and RCA1, whose products are essential for assembly of mitochondrial inner membrane enzyme complexes, suggests a related role for MBA1. The predicted translation product is a 30 kDa hydrophilic protein with a putative mitochondrial targeting sequence and no homology to any sequence in protein or EST databases. Gene disruption leads to a partial respiratory growth defect, which is more pronounced at temperatures above 30 degrees C. Concomitantly, amounts of cytochromes b and aa3 are reduced. A C-terminal c-myc-tagged MBA1 gene product is functional and is found associated with the mitochondrial inner membrane, from which it can he extracted by carbonate, but not by high salt. These observations give further support to a role of MBA1 in assembly of the respiratory chain.     

hTom34: a novel translocase for the import of proteins into human mitochondria

Most mitochondrial proteins are nuclear encoded, synthesized on cytosolic ribosomes, and imported into the mitochondria. We have identified and characterized a 309 amino acid human protein with a molecular weight of 34 kDa that functions as a subunit of the translocase for the import of such proteins. hTom34 (34-kDa Translocase of the Outer Mitochondrial Membrane) is displayed on the surface of mitochondria and is resistant to extraction under alkaline conditions. Antibodies raised against hTom34 specifically inhibit in vitro import of the mitochondrial precursor protein preornithine transcarbamylase into mitochondria isolated from rat liver. Based on trypsin digestion experiments, the receptor has a large (27 kDa) C-terminal domain exposed to the cytosol. This novel component of the protein import machinery possesses a 62 residue motif conserved with the Tom70 family of mitochondrial receptors but otherwise appears to have no counterpart so far characterized in the mitochondria of any other species.     

Mitochondrial import of only one of three nuclear-encoded glutamine tRNAs in Tetrahymena thermophila

The mitochondrial genome of Tetrahymena does not appear to encode enough tRNAs to perform mitochondrial protein synthesis. It has therefore been proposed that nuclear-encoded tRNAs are imported into the mitochondria. T.thermophila has three major glutamine tRNAs: tRNA(Gln)(UUG), tRNA(Gln)(UUA) and tRNA(Gln)(CUA). Each of these tRNAs functions in cytosolic translation. However, due to differences between the Tetrahymena nuclear and mitochondrial genetic codes, only tRNA(Gln)(UUG) has the capacity to function in mitochondrial translation as well. Here we show that approximately 10-20% of the cellular complement of tRNA(Gln)(UUG) is present in mitochondrial RNA fractions, compared with 1% or less for the other two glutamine tRNAs. Furthermore, this glutamine tRNA is encoded only by a family of nuclear genes, the sequences of several of which are presented. Finally, when marked versions of tRNA(Gln)(UUG) and tRNA(Gln)(UUA) flanked by identical sequences are expressed in the macronucleus, only the former undergoes mitochondrial import; thus sequences within tRNA(Gln)(UUG) direct import. Because tRNA(Gln)(UUG) is a constituent of mitochondrial RNA fractions and is encoded only by nuclear genes, and because ectopically expressed tRNA(Gln)(UUG) fractionates with mitochondria like its endogenous counterpart, we conclude that it is an imported tRNA in T.thermophila.     

Functional interactions between yeast mitochondrial ribosomes and mRNA 5' untranslated leaders

Translation of mitochondrial mRNAs in Saccharomyces cerevisiae depends on mRNA-specific translational activators that recognize the 5' untranslated leaders (5'-UTLs) of their target mRNAs. We have identified mutations in two new nuclear genes that suppress translation defects due to certain alterations in the 5'-UTLs of both the COX2 and COX3 mRNAs, indicating a general function in translational activation. One gene, MRP21, encodes a protein with a domain related to the bacterial ribosomal protein S21 and to unidentified proteins of several animals. The other gene, MRP51, encodes a novel protein whose only known homolog is encoded by an unidentified gene in S. kluyveri. Deletion of either MRP21 or MRP51 completely blocked mitochondrial gene expression. Submitochondrial fractionation showed that both Mrp21p and Mrp51p cosediment with the mitochondrial ribosomal small subunit. The suppressor mutations are missense substitutions, and those affecting Mrp21p alter the region homologous to E. coli S21, which is known to interact with mRNAs. Interactions of the suppressor mutations with leaky mitochondrial initiation codon mutations strongly suggest that the suppressors do not generally increase translational efficiency, since some alleles that strongly suppress 5'-UTL mutations fail to suppress initiation codon mutations. We propose that mitochondrial ribosomes themselves recognize a common feature of mRNA 5'-UTLs which, in conjunction with mRNA-specific translational activation, is required for organellar translation initiation.     

Activity and cellular location in Saccharomyces cerevisiae of chimeric mouse/yeast and Bacillus subtilis/yeast ferrochelatases

We have constructed a series of chimeric yeast/mouse and yeast/Bacillus subtilis ferrochelatase genes in order to investigate domains of the ferrochelatase that are important for activity and/or association with the membrane. These genes were expressed in a Saccharomyces cerevisiae mutant in which the endogenous ferrochelatase gene (HEM15) had been deleted, and the phenotypes of the transformants were characterized. Exchanging the approximately 40-amino-acid C-terminus between the yeast and mouse ferrochelatases caused a total loss of activity and the hybrid proteins were unstable when overproduced in Escherichia coli. The water-soluble ferrochelatase of B. subtilis did not complement the yeast mutant, although a large amount of active protein accumulated in the cytosol. Addition of the N-terminal leader sequence of yeast ferrochelatase to the B. subtilis enzyme targeted the fusion protein to mitochondria, but both the precursor and the mature forms of the enzyme were inactive in vivo and had residual activity when measured in vitro. An internal approximately 45-amino-acid segment located at the N-terminus of yeast ferrochelatase was identified, which, when replaced with the corresponding 30-amino-acid segment of the B. subtilis enzyme, caused the yeast enzyme to be located in the mitochondrial matrix as a soluble protein. The fusion protein was inactive in vivo and had residual activity in vitro. We speculate that this segment, which shows the greatest variability between species, is responsible for the association of the enzyme with the membrane.     

Reduced dosage of genes encoding ribosomal protein S18 suppresses a mitochondrial initiation codon mutation in Saccharomyces cerevisiae

A yeast mitochondrial translation initiation codon mutation affecting the gene for cytochrome oxidase subunit III (COX3) was partially suppressed by a spontaneous nuclear mutation. The suppressor mutation also caused cold-sensitive fermentative growth on glucose medium. Suppression and cold sensitivity resulted from inactivation of the gene product of RPS18A, one of two unlinked genes that code the essential cytoplasmic small subunit ribosomal protein termed S18 in yeast. The two S18 genes differ only by 21 silent substitutions in their exons; both are interrupted by a single intron after the 15th codon. Yeast S18 is homologous to the human S11 (70% identical) and the Escherichia coli S17 (35% identical) ribosomal proteins. This highly conserved family of ribosomal proteins has been implicated in maintenance of translational accuracy and is essential for assembly of the small ribosomal subunit. Characterization of the original rps18a-1 missense mutant and rps18a delta and rps18b delta null mutants revealed that levels of suppression, cold sensitivity and paromomycin sensitivity all varied directly with a limitation of small ribosomal subunits. The rps18a-1 mutant was most affected, followed by rps18a delta then rps18b delta. Mitochondrial mutations that decreased COX3 expression without altering the initiation codon were not suppressed. This allele specificity implicates mitochondrial translation in the mechanism of suppression. We could not detect an epitope-tagged variant of S18 in mitochondria. Thus, it appears that suppression of the mitochondrial translation initiation defect is caused indirectly by reduced levels of cytoplasmic small ribosomal subunits, leading to changes in either cytoplasmic translational accuracy or the relative levels of cytoplasmic translation products.     

A novel DNA-binding protein bound to the mitochondrial inner membrane restores the null mutation of mitochondrial histone Abf2p in Saccharomyces cerevisiae

The yeast mitochondrial HMG-box protein, Abf2p, is essential for maintenance of the mitochondrial genome. To better understand the role of Abf2p in the maintenance of the mitochondrial chromosome, we have isolated a multicopy suppressor (YHM2) of the temperature-sensitive defect associated with an abf2 null mutation. The function of Yhm2p was characterized at the molecular level. Yhm2p has 314 amino acid residues, and the deduced amino acid sequence is similar to that of a family of mitochondrial carrier proteins. Yhm2p is localized in the mitochondrial inner membrane and is also associated with mitochondrial DNA in vivo. Yhm2p exhibits general DNA-binding activity in vitro. Thus, Yhm2p appears to be novel in that it is a membrane-bound DNA-binding protein. A sequence that is similar to the HMG DNA-binding domain is important for the DNA-binding activity of Yhm2p, and a mutation in this region abolishes the ability of YHM2 to suppress the temperature-sensitive defect of respiration of the abf2 null mutant. Disruption of YHM2 causes a significant growth defect in the presence of nonfermentable carbon sources such as glycerol and ethanol, and the cells have defects in respiration as determined by 2,3,5,-triphenyltetrazolium chloride staining. Yhm2p may function as a member of the protein machinery for the mitochondrial inner membrane attachment site of mitochondrial DNA during replication and segregation of mitochondrial genomes.     

A nonsense mutation (G15059A) in the cytochrome b gene in a patient with exercise intolerance and myoglobinuria

We describe a new mitochondrial DNA mutation in the cytochrome b gene in a patient presenting with progressive exercise intolerance and myoglobinuria associated with complex III deficiency in muscle. The point mutation results in the replacement of a glycine at amino acid position 190 with a stop codon. This change predicts premature termination of translation, leading to a truncated protein missing 244 amino acids at the C-terminus of cytochrome b. The mutation fulfills all the accepted criteria for pathogenicity, suggesting that this is the primary cause of the myopathy in the patient.