Integrity of mitochondria in a mammalian cell mutant defective in mitochondrial protein synthesis.

A defect in mitochondrial protein synthesis has previously been identified in the respiration-deficient Chinese hamster lung fibroblast mutant V79-G7. The present work extends the characterization of this mutant. A more sensitive analysis has shown that mutant mitochondria synthesize all mitochondrially encoded peptides, but in significantly reduced amounts. This difference is also seen when isolated mitochondria are tested for in vitro protein synthesis. To distinguish between a defect in the translational machinery and a defect in the transcription of mitochondrial DNA, we investigated the synthesis of the 16S and 12S mitochondrial rRNA species and found them to be made in normal amounts in G7 mitochondria. These rRNA species appear to be assembled into subunits whose sedimentation behavior is virtually indistinguishable from that of the wild-type subunits. We also examined the consequences of the defect in mitochondrial protein synthesis on mutant cells and their mitochondria-utilizing techniques of electron microscopy, two-dimensional gel electrophoresis and immunochemical analysis. G7 mitochondria have a characteristic ultrastructure distinguished by predominantly tubular cristae, but the overall biochemical composition of mitochondrial membrane and matrix fractions appears essentially unaltered except for the absence of a few characteristic peptides. Specifically, we identify the absence of two mitochondrially encoded subunits of cytochrome c oxidase on two-dimensional gels and demonstrate a drastic reduction of both cytoplasmically and mitochondrially synthesized subunits of enzyme in immunoprecipitates of G7 mitochondria.

Cytoplasmic inheritance of oligomycin resistance in Chinese hamster ovary cells.

Oligomycin-resistant clones were isolated from Chinese hamster ovary cells by treatment of cells with ethidium bromide, followed by mutagenesis with ethylmethane sulfonate and selection in oligomycin. One clone (Olir 8.1) was chosen for further study. Olir 8.1 cells grow with doubling time similar to that of wild-type cells, whether grown in the presence or absence of drug (doubling time of 13-14 h). In plating efficiency experiments, Olir 8.1 cells are approximately 100-fold more resistant to oligomycin than are wild-type cells. There is approximately a 32-fold increase in the resistance to inhibition by oligomycin of the mitochondrial ATPase from Olir 8.1 cells. The electron transport chain is functional in Olir 8.1 cells. Oligomycin resistance is stable in the absence of selective pressure. There is little or no cross-resistance of Olir 8.1 cells to venturicidin and dicyclohexylcarbodiimide, other inhibitors of the mitochondrial ATPase, or to chloramphenicol, an inhibitor of mitochondrial protein synthesis. Oligomycin resistance is dominant in hybrids between Olir 8.1 cells and wild-type cells. Fusions of enucleated Olir 8.1 cells with sensitive cells and characterization of the resulting "cybrid" clones indicates that oligomycin resistance in Olir 8.1 cells is cytoplasmically inherited.

Isopycnic centrifugation of chromatin in renografin solutions.

Solutions of Renografin (30-60%) can be centrifuged to form density gradients in the range from 1.0 g/cm(3) to 1.4 g/cm(3) or, alternatively, preformed gradients can be made which under appropriate conditions of centrifugation have an indefinite stability. Such solutions have a low viscosity and a relatively low ionic strength. The density of DNA in such solutions is surprisingly low ( approximately 1.14 g/cm(3)). Crude chromatin can be sedimented to an equilibrium position in such gradients, corresponding to a density of 1.2(4) g/cm(3), or slightly lower, depending on the method of preparation. The complex is shown to contain DNA, RNA, protein, and possibly some lipoprotein. Most of the RNA can be removed with RNase without any significant effect on the density of the chromatin.

Biochemical and genetic characterization of three hamster cell mutants resistant to diphtheria toxin.

We describe here three different hamster cell mutants which are resistant to diphtheria toxin and which provide models for investigating some of the functions required by the toxin inactivates elongation factor 2 (EF-2). Cell-free extracts from mutants Dtx(r)-3 was codominant. The evidence suggests that the codominant phenotype is the result of a mutation in a gene coding for EF-2. The recessive phenotype might arise by alteration of an enzyme which modifies the structure of EF-2 so that it becomes a substrate for reaction with the toxin. Another mutant, Dtx(r)-2, contained EF-2 that was sensitive to the toxin and this phenotype was recessive. Pseudomonas aeruginosa exotoxin is known to inactivate EF-2 as does diphtheria toxin and we tested the mutants for cross-resistance to pseudomonas exotoxin. Dtx(r)-1 and Dtx(r)-3 were cross-resistant while Dtx(r)-2 was not. It is known that diphtheria toxin does not penetrate to the cytoplasm of mouse cells and that these cell have a naturally occurring phenotype of diphtheria toxin resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance of the hybrid cells to diphtheria toxin. Intraspecies hybrids containing the genome of mutants Dtx(r)-1 and Dtx(r)-3 had some resistance while those formed with Dtx(r)-2 were as sensitive as hybrids derived from fusions between wild-type hamster cells and mouse 3T3 cells.

Investigations of the potential effects of phosphorylation of the MWFE and ESSS subunits on complex I activity and assembly.

There have been several reports on the phosphorylation of various subunits of NADH-ubiquinone oxidoreductase (complex I) in mammalian mitochondria. The effects of phosphorylation on assembly or activity of these subunits have not been investigated directly. The cAMP-dependent phosphorylation of the MWFE and ESSS subunits in isolated bovine heart mitochondria has been recently reported. We have investigated the significance of potential phosphorylation of these two subunits in complex I assembly and function by mutational analysis of the phosphorylation sites. Chinese hamster mutant cell lines missing either the MWFE or the ESSS subunits were transfected and complemented with the corresponding wild type and mutant cDNAs made by site-directed mutagenesis. In MWFE the serine 55 was substituted by alanine, glutamate, glutamine, and aspartate (S55A, S55E, S55Q, and S55D, respectively). The glutamate substitutions might be expected to mimic the phosphorylated state of the protein. With the exception of the MWFE(S55A) mutant protein the assembly of complex I was completely blocked, and no activity could be detected. Various substitutions in the ESSS protein (S2A, S2E, S8A, S8E, T21A, T21E, S30A, S30E) appeared to cause lower levels of mature protein and a significantly reduced complex I activity measured polarographically. The ESSS (S2/8A) double mutant protein caused a complete failure to assemble. These mutational analyses suggest that if phosphorylation occurs in vivo, the effects on complex I activity are significant.

Temperature-sensitive Chinese hamster fibroblast mutant with a defect in RNA metabolism.

We describe a new temperature-sensitive mutant of Chinese hamster cell fibroblasts. After a shift to the nonpermissive temperature of 40.5 degrees C, the rates of DNA, RNA, and protein synthesis declined rapidly (to < or = 50% within 12 h) and the progression of unsynchronized cells through the cell cycle was affected. We believe that DNA synthesis came to a halt after a short time, because cells no longer entered the S phase. The decrease in protein synthesis at 40.5 degrees C was shown to be a consequence of a decrease in the number of polysomes, whereas free 80S ribosomes accumulated. We concluded that the components of the protein biosynthetic machinery were intact (ribosomes and soluble factors), but synthesis was limited by a shortage of mRNA. The decline in mRNA production had a significant effect on the synthesis of proteins (e.g., heat shock proteins) translated from short-lived messages. We observed that both polyadenylated and nonpolyadenylated RNA syntheses declined at 40.5 degrees C, whereas the synthesis of small RNAs (4 to 5S) was less reduced. The argument is made that the temperature-sensitive phenotype is the result of a defect affecting mRNA synthesis.

Control of mRNA turnover as a mechanism of glucose repression in Saccharomyces cerevisiae.

We have examined the expression of the gene encoding the iron-protein subunit (Ip) of succinate dehydrogenase in Saccharomyces cerevisiae. The gene had been cloned by us and shown to be subject to glucose regulation (A. Lombardo, K. Carine, and I. E. Scheffler, J. Biol. Chem. 265:10419-10423, 1990). We discovered that a significant part of the regulation of the Ip mRNA levels by glucose involves the regulation of the turnover rate of this mRNA. In the presence of glucose, the half-life appears to be less than 5 min, while in glycerol medium, the half-life is greater than 60 min. The gene is also regulated transcriptionally by glucose. The upstream promoter sequence appeared to have four regulatory elements with consensus sequences shown to be responsible for the interaction with the HAP2/3/4 regulatory complex. A deletion analysis has shown that the two distal elements are redundant. These measurements were carried out by Northern (RNA) analyses of Ip mRNA transcripts as well as by assays of beta-galactosidase activity in cells carrying constructs of the Ip promoter linked to the lacZ coding sequence. These observations on the regulation of mRNA stability were also extended to the mRNA of the flavoprotein subunit of succinate dehydrogenase and in some experiments of iso-1-cytochrome c.

Glucose-dependent turnover of the mRNAs encoding succinate dehydrogenase peptides in Saccharomyces cerevisiae: sequence elements in the 5' untranslated region of the Ip mRNA play a dominant role.

We have demonstrated previously that glucose repression of mitochondrial biogenesis in Saccharomyces cerevisiae involves the control of the turnover of mRNAs for the iron protein (Ip) and flavoprotein (Fp) subunits of succinate dehydrogenase (SDH). Their half-lives are > 60 min in the presence of a nonfermentable carbon source (YPG medium) and < 5 min in glucose (YPD medium). This is a rare example in yeast in which the half-lives are > 60 min in the presence of a nonfermentable carbon source (YPG medium) and < 5 min in glucose (YPD medium). This is a rare example in yeast in which the half-life of an mRNA can be controlled by manipulating external conditions. In our current studies, a series of Ip transcripts with internal deletions as well as chimeric transcripts with heterologous sequences (internally or at the ends) have been examined, and we established that the 5'-untranslated region (5' UTR) of the Ip mRNA contains a major determinant controlling its differential turnover in YPG and YPD. Furthermore, the 5' exonuclease encoded by the XRN1 gene is required for the rapid degradation of the Ip and Fp mRNAs upon the addition of glucose. In the presence of cycloheximide the nucleolytic degradation of the Ip mRNA can be slowed down by stalled ribosomes to allow the identification of intermediates. Such intermediates have lost their 5' ends but still retain their 3' UTRs. If protein synthesis is inhibited at an early initiation step by the use of a prt1 mutation (affecting the initiation factor eIF3), the Ip and Fp mRNAs are very rapidly degraded even in YPG. Significantly, the arrest of translation by the introduction of a stable hairpin loop just upstream of the initiation codon does not alter the differential stability of the transcript in YPG and YPD. These observations suggest that a signaling pathway exists in which the external carbon source can control the turnover of mRNAs of specific mitochondrial proteins. Factors must be present that control either the activity or more likely the access of a nuclease to the select mRNAs. As a result, we propose that a competition between initiation of translation and nuclease action at the 5' end of the transcript determines the half-life of the Ip mRNA.

Molecular genetics of succinate:quinone oxidoreductase in eukaryotes.

Succinate:quinone oxidoreductase is a membrane-associated complex in mitochondria, often referred to as complex II, based on the fractionation scheme developed by Y. Hatefi and colleagues. It consists of four peptides, two of which are integral membrane proteins (15 and 12-13 kDa, respectively) and two others that are peripheral membrane proteins, i.e., a flavoprotein (Fp, 70 kDa) and an iron-protein (Ip, 27 kDa). The mature, functional complex contains a cytochrome in association with the membrane proteins, a flavin linked covalently to the largest peptide, and three iron-sulfur clusters in the 27-kDa subunit. The present review touches only briefly on the biochemical and biophysical properties of this complex. Instead, the focus is on the molecular-genetic studies that have become possible since the first genes from eukaryotes were cloned in 1989. The evolutionary conservation of the amino acid sequence of both the Fp and the Ip peptides has facilitated the cloning of these genes from a large variety of eukaryotic organisms by PCR-based methods. The review addresses questions related to the regulation of the expression of these genes, with an emphasis on mammals and yeast, for which most of the information is available. Four different genes have to be co-ordinately regulated. Transcriptional as well as posttranscriptional regulatory mechanisms have been observed in diverse organisms. Intriguing observations have been made in studies of this enzyme during the life cycle of organisms existing alternately under aerobic and anaerobic conditions. Naturally occurring or induced mutations in these genes have shed light on several questions related to the assembly of this complex, and on the relationship between structure and function. Four different peptides are imported into the mitochondria. They have to be modified, folded, and assembled. The stage is set for the exploration of highly specific changes introduced by site-directed mutagenesis. Until recently the genes were believed to be exclusively nuclear in all eukaryotes, but exceptions have since been found. This finding has relevance in the discussion of the evolution of mitochondria from prokaryotes. A highly conserved set of genes is found in prokaryotes, and some informative comparisons on gene organization and expression in prokaryotes and eukaryotes have been included.

Combined and individual mitochondrial HSP60 and HSP10 expression in cardiac myocytes protects mitochondrial function and prevents apoptotic cell deaths induced by simulated ischemia-reoxygenation.

BACKGROUND: The mitochondrial heat-shock proteins HSP60 and HSP10 form a mitochondrial chaperonin complex, and previous studies have shown that their increased expression exerts a protective effect against ischemic injury when cardiac myocytes are submitted to simulated ischemia. The more detailed mechanisms by which such a protective effect occurs are currently unclear. We wanted to determine whether HSP60 and HSP10 could exert a protection against simulated ischemia and reoxygenation (SI/RO)-induced apoptotic cell death and whether such protection results from decreased mitochondrial cytochrome c release and caspase-3 activation and from the preservation of ATP levels by preservation of the electron transport chain complexes. In addition, we explored whether increased expression of HSP60 or HSP10 by itself exerts a protective effect. METHODS AND RESULTS: We overexpressed HSP60 and HSP10 together or separately in rat neonatal cardiac myocytes using an adenoviral vector and then subjected the myocytes to SI/RO. Cell death and apoptosis in myocytes were quantified by parameters such as enzyme release, DNA fragmentation, and caspase-3 activation. Overexpression of the combination of HSP60 and HSP10 and of HSP60 or HSP10 individually protected myocytes against apoptosis. This protection is accompanied by decreases in mitochondrial cytochrome c release and in caspase-3 activity and increases in ATP recovery and activities of complex III and IV in mitochondria after SI/RO. CONCLUSIONS: These results suggest that mitochondrial chaperonins HSP60 and HSP10 in combination or individually play an important role in maintaining mitochondrial integrity and capacity for ATP generation, which are the crucial factors in determining survival of cardiac myocytes undergoing ischemia/reperfusion injury.

Localization of ornithine decarboxylase in mutant CHO cells that overproduce the enzyme. Differences between the intracellular distribution of monospecific ornithine decarboxylase antibodies and radiolabeled alpha-difluoromethylornithine.

The intracellular localization of ornithine decarboxylase (ODC), a key enzyme in polyamine synthesis and cell growth, is a matter of present debate. Using two independent methods of analysis, we have attempted to determine the actual distribution of ODC in a mammalian cell. To overcome the problem of a normally very low cellular ODC content, we have used ODC overproducing mutant CHO cells. These mutant cells exhibit a 10-fold higher ODC activity than do the wild type cells. The localization of ODC protein in exponentially growing cells, was determined by indirect immunofluorescence microscopy (permeabilized whole-cell preparations and 1 micron sections), using a monospecific ODC antibody. The intracellular localization of catalytically active ODC was determined by light and electron microscope autoradiography following pulselabeling of cells with alpha-difluoromethyl(5-3H)ornithine (3H-DFMO) at the time of peak ODC activity. alpha-Difluoromethylornithine (DFMO) is an enzyme-activated irreversible inhibitor of ODC and binds covalently to the active enzyme. The specificity of this reaction in the cell was ascertained by immunoprecipitation of 3H-DFMO-labeled ODC. ODC (as determined by both methods) was present in all the cells of a serum-stimulated monolayer culture. The highest concentration of ODC protein and of catalytically active ODC was observed in the smallest and most rapidly proliferating cells. Polyploid and multinuclear cells always exhibited the lowest concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Mitochondria make a come back.

This review attempts to summarize our present state of knowledge of mitochondria in relation to a number of areas of biology, and to indicate where future research might be directed. In the evolution of eukaryotic cells mitochondria have for a long time played a prominent role. Nowadays their integration into many activities of a cell, and their dynamic behavior as subcellular organelles within a cell and during cell division are a major focus of attention. The crystal structures of the major complexes of the electron transport chain (except complex I) have been established, permitting increasingly detailed analyses of the important mechanism of proton pumping coupled to electron transport. The mitochondrial genome and its replication and expression are beginning to be understood in considerable detail, but more questions remain with regard to mutations and their repair, and the segregation of the mtDNA in oogenesis and development. Much emphasis and a large effort have recently been devoted to understand the role of mitochondria in programmed cell death (apoptosis). The understanding of their central role in mitochondrial diseases is a major achievement of the past decade. Finally, various drugs have traditionally played a part in understanding biochemical mechanisms within mitochondria; the repertoire of drugs with novel and interesting targets is expanding.

Control of mRNA turnover as a mechanism of glucose repression in Saccharomyces cerevisiae.

The phenomenon of glucose repression in yeast is concerned with the repression of a large number of genes when glucose is an abundant carbon source and almost all of the energy requirements of the cell can be satisfied from glycolysis. Prominent among the repressed genes are those encoding mitochondrial proteins required for respiration and oxidative phosphorylation. Past studies have characterized a pathway by which a signal generated from extracellular glucose is transmitted to the nucleus. The ultimate outcome is the repression of transcription of numerous genes, but also the induction of a limited number of others. The emphasis has been almost exclusively on transcriptional control mechanisms. A discovery made originally with the transcript of the SDH2 gene prompted an investigation of post-transcriptional mechanisms, and more specifically a study of the turnover rate of this mRNA in the absence and presence of glucose. SDH2 mRNA has a very short half-life in medium with glucose (YPD) and a significantly longer half-life in medium with glycerol (YPG). Experimental evidence and recent progress in understanding of (1) mRNA turnover in yeast and (2) initiation of translation on the 5' untranslated region of mRNAs, lead to a working hypothesis with the following major features: the carbon source, via a signaling pathway involving kinase/phosphatase activities, controls the rate of initiation, and thus influences a competition between eukaryotic initiation factors (prominently eIF4E, eIF4G, eIF3) binding to the capped mRNA and a decapping activity (DCP1) which is one of the rate limiting activities in the turnover of such mRNAs.

Characterization of the gene encoding the iron-sulfur protein subunit of succinate dehydrogenase from Drosophila melanogaster.

The iron-sulfur protein (Ip) subunit of succinate dehydrogenase (and complex II of the electron transport chain) is highly conserved in evolution [Gould et al., Proc. Natl. Acad. Sci. USA 86 (1989) 1934-1938]. We have cloned the Drosophila melanogaster Ip-encoding gene (SdhB) by genomic library screening using the human Ip-encoding cDNA as a probe at low stringency. A 2.7-kb fragment containing SdhB has been sequenced and shown to comprise the entire transcribed region and more than 900 bp of promoter region. The gene contains three exons and two small introns of 272 and 56 nt, respectively, and is transcribed into an mRNA of 1205 nt (plus poly(A) tail). The deduced amino-acid (aa) sequence shows strong similarities with Ip peptides from Escherichia coli, yeasts, plants and mammals, with 100% aa identity around the three Cys clusters which form the non-heme iron-sulfur centers. In situ hybridization on polytene chromosomes maps SdhB to band 42D 1-5 on the right arm of the second chromosome next to the centromere. Developmental and tissue-specific Northern blots show a single transcript of 1.3 kb in all tissues. However, its abundance varies during development and in the major body segments of the adult fly. Pupae have very low levels of transcript, in contrast to larvae. It is most abundant in the adult thorax and low in abdominal tissues.

Characterization of the human SDHC gene encoding of the integral membrane proteins of succinate-quinone oxidoreductase in mitochondria.

Complex II of mitochondria contains succinate dehydrogenase and subunits to link this enzyme directly to the inner mitochondrial membrane. The four peptides of this complex are the flavoprotein (Fp) and iron-sulfur protein (Ip) of the dehydrogenase, and two integral membrane proteins referred to as C(II-3) and C(II-4). Their respective genes in mammals are SDHA, SDHB, SDHC and SDHD) in order of decreasing molecular weights of the peptides. In this paper we describe the identification of two pseudogenes and the complete characterization and mapping of the active SDHC gene in humans. The active gene, encoding a small peptide of 15.5 kDa, has six exons and five introns extending over 35 kb. It has been mapped at position 1q21, adjacent to the pericentric heterochromatin on the long arm of chromosome 1. Approximately I kb of the promoter region has also been sequenced and examined for sequence motifs suggesting the binding of known transcription factors. Several potential sites for the nuclear respiratory factors NRF-1 and NRF-2 were identified.

Structural organization of the gene encoding the human iron-sulfur subunit of succinate dehydrogenase.

The iron-sulfur protein (Ip) subunit of succinate dehydrogenase (SDH and complex II) of the respiratory chain is highly conserved in evolution [Gould et al., Proc. Natl. Acad. Sci. USA 86 (1989) 1934-1938]. We have cloned the entire human Ip cDNA, as well as the Ip-encoding gene (SDH-B) from two genomic human libraries. The cDNA contains a coding sequence of 840 nt, flanked by a 5'-UTR of 133 nt and a 3'-UTR of 123 nt. The entire transcript is encoded by eight exons within approx. 40 kb. The seven introns range in size from 0.75 kb to > 11 kb, and they appear to be of the 'late' intron class. Approx. 5 kb of upstream sequence was also cloned, and approx. 2.4 kb of the promoter region were sequenced and analyzed for consensus elements binding potential transcription factors and transcriptional activators.