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1.
Inactivation of Saccharomyces cerevisiae OGG1 DNA repair gene leads to an increased frequency of mitochondrial mutants 总被引:3,自引:0,他引:3
The OGG1 gene encodes a highly conserved DNA glycosylase that repairs oxidized guanines in DNA. We have investigated the in vivo function of the Ogg1 protein in yeast mitochondria. We demonstrate that inactivation of ogg1 leads to at least a 2-fold increase in production of spontaneous mitochondrial mutants compared with wild-type. Using green fluorescent protein (GFP) we show that a GFP–Ogg1 fusion protein is transported to mitochondria. However, deletion of the first 11 amino acids from the N-terminus abolishes the transport of the GFP–Ogg1 fusion protein into the mitochondria. This analysis indicates that the N-terminus of Ogg1 contains the mitochondrial localization signal. We provide evidence that both yeast and human Ogg1 proteins protect the mitochondrial genome from spontaneous, as well as induced, oxidative damage. Genetic analyses revealed that the combined inactivation of OGG1 and OGG2 [encoding an isoform of the Ogg1 protein, also known as endonuclease three-like glycosylase I (Ntg1)] leads to suppression of spontaneously arising mutations in the mitochondrial genome when compared with the ogg1 single mutant or the wild-type. Together, these studies provide in vivo evidence for the repair of oxidative lesions in the mitochondrial genome by human and yeast Ogg1 proteins. Our study also identifies Ogg2 as a suppressor of oxidative mutagenesis in mitochondria. 相似文献
2.
It has previously been shown that the mitochondrial DNA (mtDNA) of Saccharomyces cerevisiae becomes thermosensitive due to the inactivation of the mitochondrial DNA helicase gene, PIF1. A suppressor of this thermosensitive phenotype was isolated from a wild-type plasmid library by transforming a pif1 null strain to growth on glycerol at the non-permissive temperature. This suppressor is a nuclear gene encoding a 135 amino acid protein that is itself essential for mtDNA replication; cells lacking this gene are totally devoid of mtDNA. We therefore named this gene RIM1 for replication in mitochondria. The primary structure of the RIM1 protein is homologous to the single-stranded DNA binding protein (SSB) from Escherichia coli and to the mitochondrial SSB from Xenopus laevis. The mature RIM1 gene product has been purified from yeast extracts using a DNA unwinding assay dependent upon the DNA helicase activity of SV40 T-antigen. Direct amino acid sequencing of the protein reveals that RIM1 is a previously uncharacterized SSB. Antibodies against this purified protein localize RIM1 to mitochondria. The SSB encoded by RIM1 is therefore an essential component of the yeast mtDNA replication apparatus. 相似文献
3.
4.
The small nuclear gene SOM1 of Saccharomyces cerevisiae was isolated as a multicopy suppressor of a mutation in the IMP1 gene, which encodes the mitochondrial inner membrane peptidase subunit 1 (Imp1). Analysis revealed that Som1 and Imp1 are components of a mitochondrial protein export system, and interaction between these two proteins is indicated by the genetic suppression data. Here we describe the identification of a gene from Kluyveromyces lactis, which restores respiratory function to a S. cerevisiae SOM1 deletion mutant at 28°?C. The sequence of the K. lactis gene predicts a protein product of 8.1-kDa, comprising 71 amino acid residues, with a putative mitochondrial signal sequence at its N-terminus. The protein is 50% identical to its S.cerevisiae counterpart. The expression pattern of a homologous sequence in Leishmania major suggests a more general role for SOM1 in mitochondrial biogenesis and protein sorting. The various Som1 proteins exhibit a highly conserved region and a remarkable pattern of cysteine residues. A protein of the expected size was transcribed and translated in vitro. The Som1 protein was detected in fractions of S. cerevisiae enriched for mitochondria and found to be associated with the inner mitochondrial membrane. 相似文献
5.
Eric Van Dyck Bernhard Jank Antonella Ragnini Rudolf J. Schweyen Claire Duyckaerts Francis Sluse Françoise Foury 《Molecular & general genetics : MGG》1995,246(4):426-436
The PIF1 and MRS2 gene products have previously been shown to be essential for mitochondrial DNA maintenance at elevated temperatures and mitochondrial group II intron splicing, respectively, in the yeast Saccharomyces cerevisiae. A multicopy suppressor capable of rescuing the respiratory deficient phenotype associated with null alleles of either gene has been isolated. This suppressor is a nuclear gene that was called RIM2/MRS12. The RIM2/MRS12 gene encodes a predicted protein of 377 amino acids that is essential for mitochondrial DNA metabolism and proper cell growth. Inactivation of this gene causes the total loss of mitochondrial DNA and, compared to wild-type rhoo controls, a slow-growth phenotype on media containing glucose. Analysis of the RIM2/MRS12 protein sequence suggests that RIM2/MRS12 encodes a novel member of the mitochondrial carrier family. In particular, a typical triplicate structure, where each repeat consists of two putative transmembrane segments separated by a hydrophilic loop, can be deduced from amino acid sequence comparisons and the hydropathy profile of RIM2/MRS12. Antibodies directed against the aminoterminus of RIM2/MRS12 detect this protein in mitochondria. The function of the RIM2/MRS12 protein and the substrates it might transport are discussed. 相似文献
6.
Isolation of a gene encoding a mitochondrial HSP70 protein from Schizosaccharomyces pombe 总被引:2,自引:0,他引:2
We have isolated cDNA and genomic clones encoding a mitochondrial HSP70 protein from Schizosaccharomyces pombe. Nucleotide sequence analysis indicates that the encoded protein is homologous to the HSP70s of other organisms. The highest degree of amino acid conservation is with the proteins encoded by the Escherichia coli dnaK gene, the SSC1 gene of Saccharomyces cerevisiae and the MTP70 gene of Trypanosoma cruzi, the latter two having recently been shown to be located in the mitochondria. Western-blot analysis with immunoglobulin G raised against a peptide corresponding to the C terminus of the SSP1 protein indicates a 70-kDa protein which is associated with the mitochondria. 相似文献
7.
The small nuclear gene SOM1 of Saccharomyces cerevisiae was isolated as a multicopy suppressor of a mutation in the IMP1 gene, which encodes the mitochondrial inner membrane peptidase subunit 1 (Imp1). Analysis revealed that Som1 and Imp1 are
components of a mitochondrial protein export system, and interaction between these two proteins is indicated by the genetic
suppression data. Here we describe the identification of a gene from Kluyveromyces lactis, which restores respiratory function to a S. cerevisiae SOM1 deletion mutant at 28° C. The sequence of the K. lactis gene predicts a protein product of 8.1-kDa, comprising 71 amino acid residues, with a putative mitochondrial signal sequence
at its N-terminus. The protein is 50% identical to its S.cerevisiae counterpart. The expression pattern of a homologous sequence in Leishmania major suggests a more general role for SOM1 in mitochondrial biogenesis and protein sorting. The various Som1 proteins exhibit a highly conserved region and a remarkable
pattern of cysteine residues. A protein of the expected size was transcribed and translated in vitro. The Som1 protein was detected in fractions of S. cerevisiae enriched for mitochondria and found to be associated with the inner mitochondrial membrane.
Received: 22 July 1997 / Accepted: 27 October 1997 相似文献
8.
Uracil-DNA glycosylase-deficient yeast exhibit a mitochondrial mutator phenotype 总被引:1,自引:0,他引:1
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Mutations in mitochondrial DNA (mtDNA) have been reported in cancer and are involved in the pathogenesis of many mitochondrial diseases. Uracil-DNA glycosylase, encoded by the UNG1 gene in Saccharomyces cerevisiae, repairs uracil in DNA formed due to deamination of cytosine. Our study demonstrates that inactivation of the UNG1 gene leads to at least a 3-fold increased frequency of mutations in mtDNA compared with the wild-type. Using a Ung1p–green fluorescent protein (GFP) fusion construct, we demonstrate that yeast yUng1–GFP protein localizes to both mitochondria and the nucleus, indicating that Ung1p must contain both a mitochondrial localization signal (MLS) and a nuclear localization signal. Our study reveals that the first 16 amino acids at the N-terminus contain the yUng1p MLS. Deletion of 16 amino acids resulted in the yUng1p–GFP fusion protein being transported to the nucleus. We also investigated the intracellular localization of human hUng1p–GFP in yeast. Our data indicate that hUng1p–GFP predominately localizes to the mitochondria. Further analysis identified the N-terminal 16 amino acids as important for localization of hUng1 protein into the mitochondria. Expression of both yeast and human UNG1 cDNA suppressed the frequency of mitochondrial mutation in UNG1-deficient cells. However, expression of yUNG1 in wild-type cells increased the frequency of mutations in mtDNA, suggesting that elevated expression of Ung1p is mutagenic. An increase in the frequency of mitochondrial mutants was also observed when hUNG1 site-directed mutants (Y147C and Y147S) were expressed in mitochondria. Our study suggests that deamination of cytosine is a frequent event in S.cerevisiae mitochondria and both yeast and human Ung1p repairs deaminated cytosine in mitochondria. 相似文献
9.
A role for unsaturated fatty acids in mitochondrial movement and inheritance 总被引:14,自引:5,他引:9
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Yeast cells with the mdm2 mutation display temperature-sensitive growth and defective intracellular mitochondrial movement at the non-permissive temperature. The latter phenotype includes both an absence of mitochondrial transfer into daughter buds of mitotically growing cells and an aberrant mitochondrial distribution in cells exposed to mating pheromone. The wild-type MDM2 gene was cloned by complementation, and DNA sequence analysis revealed a large open reading frame encoding a putative protein of 58.4 kD. The predicted protein sequence is identical to that reported for the yeast OLE1 gene encoding fatty acid desaturase. Unsaturated fatty acid levels are substantially decreased in mdm2 cells after a prolonged incubation at the non-permissive temperature. The addition of oleic acid complements the temperature-sensitive growth and mitochondrial distribution defects of the mutant cells. These results indicate that mdm2 is a temperature-sensitive allele of OLE1 and demonstrate an essential role for unsaturated fatty acids in mitochondrial movement and inheritance. 相似文献
10.
The characterization of a mitochondrial acyl carrier protein isoform isolated from Arabidopsis thaliana. 总被引:3,自引:1,他引:2
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A cDNA clone was isolated from an Arabidopsis leaf cDNA library that shared a high degree of protein sequence identity with mitochondrial acyl carrier proteins (mtACPs) isolated from Neurospora crassa and bovine heart muscle. The cDNA encoded an 88-amino acid mature protein that was preceded by a putative 35-amino acid presequence. In vitro protein import studies have confirmed that the presequence specifically targets this protein into pea mitochondria but not into chloroplasts. These studies indicated that pea mitochondria were not only able to import and process the precursor protein but also possessed the ability to acylate the mature protein. The mitochondrial localization of this protein, mtACP-1, was confirmed by western blot analysis. Arabidopsis mitochondrial protein extracts contained two cross-reacting bands that comigrated with the mature mtACP-1 and acylated mtACP-1 proteins. The acylated form of mtACP-1 was approximately 4 times more abundant than the unacylated form and appeared to be localized predominantly in the mitochondrial membrane where the unacylated mtACP-1 was present mostly in the matrix fraction. A chloroplast fatty acid synthase system was used, and mtACP-1 was able to function as a cofactor for fatty acid synthesis. However, predominantly short- and medium-chain fatty acids were produced in fatty acid synthase reactions supplemented with mtACP-1, suggesting that mtACP-1 may be causing premature fatty acid chain termination. 相似文献
11.
A Miranda-Vizuete A E Damdimopoulos J R Pedrajas J A Gustafsson G Spyrou 《European journal of biochemistry》1999,261(2):405-412
We have isolated a 1918-bp cDNA from a human adrenal cDNA library which encodes a novel thioredoxin reductase (TrxR2) of 521 amino acid residues with a calculated molecular mass of 56.2 kDa. It is highly homologous to the previously described cytosolic enzyme (TrxR1), including the conserved active site CVNVGC and the FAD-binding and NADPH-binding domains. However, human TrxR2 differs from human TrxR1 by the presence of a 33-amino acid extension at the N-terminus which has properties characteristic of a mitochondrial translocation signal. Northern-blot analysis identified one mRNA species of 2.2 kb with highest expression in prostate, testis and liver. We expressed human TrxR2 as a fusion protein with green fluorescent protein and showed that in vivo it is localized in mitochondria. Removal of the mitochondrial targeting sequence abolishes the mitochondrial translocation. Finally, we determined the genomic organization of the human TrxR2 gene, which consists of 18 exons spanning about 67 kb, and its chromosomal localization at position 22q11.2. 相似文献
12.
Smith Penelope M.C. Mann Anthea J. Goggin Danica E. Atkins Craig A. 《Plant molecular biology》1998,36(6):811-820
A cDNA (VUpur5) encoding phosphoribosyl aminoimidazole (AIR) synthetase, the fifth enzyme of the de novo purine biosynthesis pathway has been isolated from a cowpea nodule cDNA library. It encodes a 388 amino acid protein with a predicted molecular mass of 40.4 kDa. The deduced amino acid sequence has significant homology with AIR synthetase from other organisms. AIR synthetase is present in both mitochondria and plastids of cowpea nodules [7]. A signal sequence encoded by the VUpur5 cDNA has properties associated with plastid transit sequences but there is no consensus cleavage site as would be expected for a plastid targeted protein. Although the signal sequence does not have the structural features of a mitochondrial targeted protein, it has a mitochondrial cleavage site motif (RX/XS) close to the predicted N-terminus of the mature protein. Southern analysis suggests that AIR synthetase is encoded by a single gene raising questions as to how the product of this gene is targeted to the two organelles. VUpur5 is expressed at much higher levels in nodules compared to other cowpea tissues and the gene is active before nitrogen fixation begins. These results suggest that products of nitrogen fixation do not play a role in the initial induction of gene expression. VUpur5 was expressed in Escherichia coli and the recombinant protein used to raise antibodies. These antibodies recognize two forms of AIR synthetase which differ in molecular size. Both forms are present in mitochondria, although the larger protein is more abundant. Only the smaller protein was detected in plastids. 相似文献
13.
Activation of a novel long-chain free fatty acid generation and export system in mitochondria of diabetic rat hearts 总被引:2,自引:0,他引:2
Gerber LK Aronow BJ Matlib MA 《American journal of physiology. Cell physiology》2006,291(6):C1198-C1207
A number of reports indicate that a long-chain free fatty acid export system may be operating in mitochondria. In this study, we sought evidence of its existence in rat heart mitochondria. To determine its potential role, we also sought evidence of its activation or inhibition in streptozotocin (STZ)-induced diabetic rat heart mitochondria. If confirmed, it could be a novel mechanism for regulation of long-chain fatty acid oxidation (FAO) in mitochondria. To obtain evidence of its existence, we tested whether heart mitochondria presented with palmitoyl-carnitine can generate and export palmitate. We found that intact mitochondria indeed generate and export palmitate. We have also found that the rates of these processes are markedly higher in STZ-diabetic rat heart mitochondria, in which palmitoyl-carnitine oxidation is also increased. Since mitochondrial thioesterase-1 (MTE-1) hydrolyzes acyl-CoA to CoA-SH + free fatty acid, and uncoupling protein-3 (UCP-3), reconstituted in liposomes, transports free fatty acids, we examined whether these proteins are also increased in STZ-diabetic rat heart mitochondria. We found that both of these proteins are indeed increased. Gene expression profile analysis revealed striking expression of mitochondrial long-chain fatty acid transport and oxidation genes, accompanying overexpression of MTE-1 and UCP-3 in STZ-diabetic rat hearts. Our findings provide the first direct evidence for the existence of a long-chain free fatty acid generation and export system in mitochondria and its activation in STZ-diabetic rat hearts in which FAO is enhanced. We suggest that its activation may facilitate, and inhibition may limit, enhancement of FAO. fatty acid oxidation; diabetes; lipotoxic cardiomyopathy; gene array 相似文献
14.
A cDNA clone encoding a mitochondrial NADH dehydrogenase from Neurospora crassa was sequenced. The total DNA sequence encompasses 2570 base pairs and contains an open reading frame of 2019 base pairs coding for a precursor polypeptide of 673 amino acid residues. The protein is encoded by a single-copy gene located to the right side of the centromere in linkage group IV of the fungal genome. The N-terminus of the precursor protein has characteristics of a mitochondrial targeting pre-sequence. The protein displays homology with mitochondrial NADH dehydrogenases from yeast. In contrast to these polypeptides, however, analysis of its primary structure revealed that it contains a well-conserved calcium-binding domain. Rabbit antiserum against the protein expressed in an heterologous system recognises a mitochondrial protein of N. crassa with an apparent molecular mass of 64 kDa. Analysis of the fungal mitochondria by swelling, digitonin fractionation and alkaline treatment indicate that the protein is located in the inner membrane of the organelles, possibly facing the matrix side. 相似文献
15.
16.
A. Harington C. J. Herbert B. Tung G. S. Getz P. P. Slonimski 《Molecular microbiology》1993,9(3):545-555
We have analysed a new gene, CEM1, from Saccharomyces cerevisiae. Inactivation of this gene leads to a respiratory-deficient phenotype. The deduced protein sequence shows strong similarities with β-keto-acyl synthases or condensing enzymes. Typically, enzymes of this class are involved in the synthesis of fatty acids or similar molecules. An analysis of the mitochondrial lipids and fatty acids shows no major difference between the wild type and deleted strains. Implying that the CBM1 gene product is not involved in the synthesis of the bulk fatty acids. Thus it Is possible that the CEM1 protein is involved in the synthesis of a specialized molecule, probably related to a fatty acid, which is essential for mitochondrial respiration. 相似文献
17.
All mRNAs on the yeast mitochondrial genome terminate at a conserved dodecamer sequence 5'-AAUAAUAUUCUU-3'. We have characterized two mutants with altered dodecamers. One contains a deletion of the dodecamer at the end of the var1 gene, and the other contains two adjacent transversions in the dodecamer at the end of the reading frame of fit1, a gene within the omega+ allele of the 21S rRNA gene. In each mutant, expression of the respective gene is blocked completely. A dominant nuclear suppressor, SUV3-1, was isolated that suppresses the var1 deletion but is without effect on the fit1 dodecamer mutations. Unexpectedly, however, we found that SUV3-1 blocks expression of the wild-type fit1 allele by blocking processing at its dodecamer. SUV3-1 has pleiotropic effects on mitochondrial gene expression, affecting RNA processing, RNA stability, and translation. Our results suggest that RNA metabolism and translation may be part of a multicomponent complex within mitochondria. 相似文献
18.
19.
Herein, we report cloning and subcellular localization of two alanine aminotransferase (ALT) isozymes, cALT and mALT, from liver of gilthead sea bream (Sparus aurata). CHO cells transfected with constructs expressing cALT or mALT as C- or N-terminal fusion with the enhanced green fluorescent protein (EGFP) showed that cALT is cytosolic, whereas mALT localized to mitochondria. Fusion of EGFP to mALT N-terminus or removal of amino acids 1-83 of mALT avoided import into mitochondria, supporting evidence that the mALT N-terminus contains a mitochondrial targeting signal. The amino acid sequence of mALT is the first reported for a mitochondrial ALT in animals. 相似文献
20.
The adenine nucleotide translocator of higher plants is synthesized as a large precursor that is processed upon import into mitochondria. 总被引:6,自引:0,他引:6
B M Winning C J Sarah P E Purdue C D Day C J Leaver 《The Plant journal : for cell and molecular biology》1992,2(5):763-773
Two maize genes and cDNAs encoding the mitochondrial adenine nucleotide translocator (ANT), a nuclear-encoded inner mitochondrial membrane carrier protein, have previously been isolated in this laboratory. Sequence analysis revealed the existence of much longer open reading frames than the corresponding fungal and mammalian ANT genes. Potato ANT cDNAs have subsequently been isolated and sequenced and alignment of the deduced plant amino acid sequences with the equivalent fungal and mammalian polypeptides indicated that the plant proteins contain N-terminal extensions. When the plant cDNA clones are expressed in vitro they direct the synthesis of precursor proteins that are specifically processed at the N-terminus upon import into isolated mitochondria. N-terminal amino acid sequence data obtained from the native proteins purified from both maize and potato mitochondria has allowed identification of the putative processing sites. Further import analysis has shown that two distinct regions of the maize precursor protein contain targeting information, the 97 amino acids at the N-terminus and the 267 C-terminal amino acids. This is the first report that provides experimental evidence that the adenine nucleotide translocator of higher plants is synthesized as a large precursor protein that is specifically cleaved upon import into mitochondria. Import of ANT into higher plant mitochondria therefore appears to be different to the corresponding process in fungal and mammalian systems where targeting of ANT to mitochondria is mediated by internal signals and there is no N-terminal processing. 相似文献