Cytochrome c mRNA levels decrease in senescent rat heart.

The concentration of mitochondria decrease in the heart as rodents age from maturity to senescence. The reason for this change is not known. One purpose of the present study was to determine if cytochrome c mRNA, representative of proteins of the inner mitochondrial membrane, decreased in the hearts of Fischer 344 rats as they aged from 12 to 24 months. Twenty-two percent less cytochrome c mRNA existed per given quantity of extracted RNA from the heart in 24-month-old rats as compared with the 12-month-old group. No change in the quantities of cardiac -actin mRNA, Ca²⁺/calmodulin protein kinase II mRNA or 18S rRNA was noted between 12- und 24-month-old hearts. Thus, the decrease in cytochrome c mRNA suggests that decreased in mRNAs for proteins of the inner mitochondrial membrane could play some role in the diminished concentration of mitochondria that exists in the senescent heart.     

Mitochondria and Apoptosis: HQ or High-Security Prison?

Whether we view the mitochondria as the headquarters for the leader of a crack suicide squad or as a prison for the leader of a militant coup, the role of the mitochondria in the apoptotic process is now well established. During apoptosis the integrity of the mitochondria is breeched, the mitochondrial transmembrane potential drops, the electron transport chain is disrupted, and proteins from the mitochondrial intermembrane space (MIS) such as cytochrome c are released into the cytosol, although not necessarily in that order. In the cytosol, cytochrome c forms part of a proteinaceous complex that directly activates caspase-9, one of the apical enzymes responsible for the dismantling of the cell. In this way a mitochondrial factor which is normally locked away from the rest of the cell can directly trigger apoptosis. The need to regulate the release of cytochrome c suggests that the mitochondria may be the decision center for whether a cell lives or dies. Various hypotheses have been formulated to explain how proteins of the MIS are released and how this process is regulated. These include the Bcl-2-regulated opening of a permeability transition pore or an increase in mitochondrial transmembrane potential followed by outer membrane rupture. It remains to be clarified which mitochondria specific events are essential for apoptosis and which are merely consequences of apoptosis.     

Age-dependent changes in glutamate oxidation by non-synaptic and synaptic mitochondria from rat brain

The oxidation of glutamate by non-synaptic and synaptic mitochondria from brains of 3-, 12- and 24-month-old rats was studied. With glutamate plus malate as substrates, non-synaptic mitochondria showed higher respiration rates than synaptic mitochondria in all the three age groups studied. The rate of oxidation of L-[1-¹⁴C] glutamate and the activities of NAD-glutamate dehydrogenase and aspartate aminotransferase were also higher in non-synaptic mitochondria compared with synaptic mitochondria in three age groups. With glutamate plus malate as substrates, a significant reduction in state 3 respiration was observed in both mitochondrial populations from 12- and 24-month-old rats compared with 3-month-old animals. Although an age-dependent decrease in the oxidation of L-[1-¹⁴C] glutamate was observed in both non-synaptic and synaptic mitochondria from aging rats, the oxidation of [1-¹⁴C]-2-oxoglutarate was unaltered in non-synaptic and synaptic mitochondria from senescent rats. The activity of NAD-glutamate dehydrogenase was decreased with age in both mitochondrial populations, whereas aspartate aminotransferase was not altered with age. The results indicate that the oxidation rate of glutamate in rat brain mitochondria is decreased during aging.     

Transport of proteins across mitochondrial membranes

The vast majority of proteins comprising the mitochondrion are encoded by nuclear genes, synthesized on ribosomes in the cytosol, and translocated into the various mitochondrial subcompartments. During this process proteins must cross the lipid membranes of the mitochondrion without interfering with the integrity or functions of the organelle. In recent years an approach combining biochemical, molecular, genetic, and morphological methodology has provided insights into various aspects of this complex process of intracellular protein sorting. In particular, a greater understanding of the molecular specificity and mechanism of targeting of mitochondrial preproteins has been reached, as a protein complex of the outer membrane which facilitates recognition and initial membrane insertion has been identified and characterized. Furthermore, pathways and components involved in the translocation of preproteins across the two mitochondrial membranes are being dissected and defined. The energetics of translocation and the processes of unfolding and folding of proteins during transmembrane transfer are closely linked to the function of a host of proteins known as heat-shock proteins or molecular chaperones, present both outside and inside the mitochondrion. In addition, the analysis of the process of folding of polypeptides in the mitochondrial matrix has allowed novel and unexpected insights into general pathways of protein folding assisted by folding factors. Pathways of sorting of proteins to the four different mitochondrial subcompartments — the outer membrane (OM), intermembrane space, inner membrane (IM) and matrix — are only partly understood and reveal an amazing complexity and variation. Many additional protein factors are involved in these latter processes, a few of which have been analyzed, such as cytochrome c heme lyase and cytochrome c ₁ heme lyase, enzymes that catalyze the covalent addition of the heme group to cytochrome c and c ₁ preproteins, and the mitochondrial processing peptidase which cleaves signal sequence after import of preproteins into the matrix. Thus, the study of transport of polypeptides through the mitochondrial membranes does not only contribute to the understanding of how biological membranes facilitate the penetration of macromolecules but also provides novel insights into the structure and function of this organelle. are being dissected and defined. The energetics of translocation and the processes of unfolding and folding of proteins during transmembrane transfer are closely linked to the function of a host of proteins known as heat-shock proteins or molecular chaperones, present both outside and inside the mitochondrion. In addition, the analysis of the process of folding of polypeptides in the mitochondrial matrix has allowed novel and unexpected insights into general pathways of protein folding assisted by folding factors. Pathways of sorting of proteins to the four different mitochondrial subcompartments — the outer membrane (OM), intermembrane space, inner membrane (IM) and matrix — are only partly understood and reveal an amazing complexity and variation. Many additional protein factors are involved in these latter processes, a few of which have been analyzed, such as cytochrome c heme lyase and cytochrome c ₁ heme lyase, enzymes that catalyze the covalent addition of the heme group to cytochrome c and c ₁ preproteins, and the mitochondrial processing peptidase which cleaves signal sequences after import of preproteins into the matrix. Thus, the study of transport of polypeptides through the mitochondrial membranes does not only contribute to the understanding of how biological membranes facilitate the penetration of macromolecules but also provides novel insights into the structure and function of this organelle.Abbreviations OM outer mitochondrial membrane - IM inner mitochondrial membrane - IMS mitochondrial intermembrane space - OMV outer membrane vesicles - AAC ADP/ATP carrier - PiC phosphate carrier - DHFR mouse cytosolic dihydrofolate reductase - F₁ subunit of F₁F₀ ^– ATPase - MPP mitochondrial processing peptidase     

Decreased Cytochrome c Oxidase Subunit VIIa in Aged Rat Heart Mitochondria: Immunocytochemistry

Aging decreases oxidative phosphorylation through cytochrome oxidase (COX) in cardiac interfibrillar mitochondria (IFM) in 24‐month old (aged) rats compared to 6‐month old adult Fischer 344 rats, whereas subsarcolemmal mitochondria (SSM) located beneath the plasma membrane remain unaffected. Immunoelectron microscopy (IEM) reveals in aged rats a 25% reduction in cardiac COX subunit VIIa in cardiac IFM, but not in SSM. In contrast, the content of subunit IV remains unchanged in both SSM and IFM, irrespective of age. These subunits are localized mainly on cristae membranes. In contrast, semi‐quantitative immunoblotting, which detects denatured protein, indicates that the content of COX VIIa is similar in IFM and SSM from both aged and adult hearts. IEM provides a sensitive method for precise localizing and quantifying specific mitochondrial proteins. The lack of immunoreaction of COX VIIa subunit by IEM in aged IFM is not explained by a reduction in protein, but rather by a masking phenomenon or by an in situ change in protein structure affecting COX activity. Anat Rec, 2011. © 2011 Wiley‐Liss, Inc.     

Influence of age on mitochondrial enzyme levels in Drosophila

The specific activity and the activity per fly of four mitochondrial enzymes did not change with ageing in male Drosophila melanogaster (Oregon R). The enzymes assayed were rotenone-insensitive NADH-cytochrome c reductase, adenylate kinase, succinate cytochrome c reductase, and malate dehydrogenase, located in the outer membrane, inner membrane space, inner membrane and matrix, respectively. The specific activity of malate dehydrogenase showed no significant change for young and old head, thorax and abdomen. We conclude that there is no specific site for ageing damage in the mitochondrion, when the enzyme activities in this study are used as an indicator. It should be noted, however, that these enzymes represent only a small percentage of the total enzymes present in mitochondria.     

Decrease of phosphorylating oxidation and increase of heat producing NADH oxidation in rat liver mitochondria during life-span prolongation of rats by calorie-restricted diet.

The influence of calorie-restricted diet, initiated at weaning, on some of the oxidative processes in liver homogenates and isolated mitochondria of 2-, 3-, 4-, 24-, 35- and 45-month-old male Wistar rats was studied in comparison with control ad libitum-fed 1-2 day-old rats and 0.5-, 1-, 2-, 3-, 4- and 24-month-old rats. It was shown that a calorie-restricted diet (at 37% of the ad libitum calorific level) did not change the rate of succinate oxidation coupled with oxidative phosphorylation in homogenates, but resulted in a decrease of succinate, glutamate plus malate and beta-hydroxybutyrate oxidation and cytochrome c-oxidase activity in isolated mitochondria without any uncoupling of oxidative phosphorylation or change in cytochrome content in the mitochondria. On the other hand, a significant increase in mitochondrial rotenone-insensitive NADH oxidation and a higher liver mass/body mass ratio in rats under the calorie-restricted diet was established. It may be considered that the activation of a heat-producing mechanism is a very important physiological function in such a condition.     

Regulation of heme metabolism during senescence: activity of several heme-containing enzymes and heme oxygenase in the liver and kidney of aging rats

The activities of several heme-containing enzymes plus succinate dehydrogenase, the content of mitochondrial cytochromes, the amount of microsomal cytochrome P-450, and the activity of heme oxygenase, the major enzyme of heme degradation, have been compared in young, mature and senescent rats. Measurements were made in liver, a tissue previously reported to have an age-related decrease in δ-aminolevulinic acid synthetase, and in kidney, a tissue previously reported to have no age-related change in this enzyme, the rate-limiting enzyme of heme biosynthesis (Paterniti, Lin and Beattie, Arch. Biochem. Biophys., 191 (1978) 792–797). The activity of cytochrome oxidase in liver mitochondria did not decrease with age while this activity in kidney mitochondria was highest in animals one year old and decreased in the two-year-olds. By contrast, succinate dehydrogenase of both kidney and liver mitochondria decreased markedly in the aging rats. No age-related change in the content of cytochromes b, c or aa₃ was observed in liver mitochondria; however, a marked age-related decrease in cytochrome aa₃ was observed in kidney mitochondria. Similarly no change in cytochrome P-450 levels was observed in either tissue obtained from aging animals. In the liver, catalase activity increased while in the kidney it decreased in senescent as compared to mature animals. Heme degradation does not decrease with age as the activity of heme oxygenase increased in both liver and kidney of two-year-old rats as compared to one-year-olds. These results suggest that the lower activity of δ-aminolevulinic acid synthetase observed in the aging rat may not be correlated with a decrease in the activity of heme-containing proteins and that the regulation of the heme pool used for the synthesis of various intracellular hemo-proteins may be complex.     

Rapamycin protects against rotenone-induced apoptosis through autophagy induction

Ubiquitin proteasome system (UPS) and autophagy lysosome pathway (ALP) are the two most important routes for degradation of aggregated/misfolded proteins. Additionally, ALP is so far the only known route to clear entire organelles, such as mitochondria. We proposed that enhancement of ALP may be beneficial for some neurodegenerative disorders, such as Parkinson's disease (PD), in which the accumulation of aggregated/misfolded proteins and the dysfunction of mitochondria are the two major pathogenesis. Mitochondrial complex I inhibitor rotenone, which causes dysfunction mitochondria and UPS, has been considered as one of the neurotoxins related to PD. In this study, rotenone-exposed human neuronal SH-SY5Y cells were used as an in vitro model for us to determine whether autophagy enhancer rapamycin could protect against rotenone-induced injury and its underlying mechanisms. The observed results showed that rapamycin alleviated rotenone-induced apoptosis, whose effects were partially blocked when autophagy related gene 5 (Atg5) was suppressed by Atg5 small interference RNA (siRNA) transfection. Additionally, the results showed that rapamycin pretreatment diminished rotenone-induced accumulation of high molecular weight ubiquitinated bands, and reduced rotenone-induced increase of cytochrome c in cytosolic fraction and decreased mitochondrial marker cytochrome oxidase subunit IV (COX IV) in mitochondrial fraction. The changes in cytochrome c and COX IV indicated that the decreased translocation of cytochrome c from mitochondria to cytosol was probably due to the turn over of entire injured mitochondria. The results that lysosome and mitochondria were colocolized within the cells pretreated with rapamycin and that the mitochondria could be found within autophagy double membrane structures further supported that the damaged mitochondria might be cleared through autophagy, which process has been termed as “mitophagy.” Our studies suggested that autophagy enhancer rapamycin is neuroprotective against rotenone-induced apoptosis through autophagy enhancement. We concluded that pharmacologically induction of autophagy by rapamycin may represent a useful therapeutic strategy as disease-modifiers in PD.     

Saccharomyces cerevisiae translational activator Cbs2p is associated with mitochondrial ribosomes

A characteristic feature of the mitochondrial expression system in Saccharomyces cerevisiae is the requirement for gene-specific translational activator proteins. Translation of mitochondrial apocytochrome b mRNA requires the nucleus-encoded proteins Cbs1p and Cbs2p. These proteins are thought to tether cytochrome b mRNA to the mitochondrial inner membrane via binding to the 5 untranslated mRNA leader. Here, we demonstrate by the use of affinity chromatography and coimmunoprecipitation that Cbs2p interacts with the mitoribosomes. We further provide evidence that the C-terminus of Cbs2p is important for ribosome association, while the N-terminal portion is essential for the formation of homomeric structures.     

Effect of Hypoxia on Mitochondrial Mass and Cytochrome Concentrations in Rat Heart and Liver during Postnatal Development

Cytochrome concentrations of rat heart and liver mitochondria were measured postnatally from newborn animals to young adults. Mitochondrial cytochrome aa₃ concentration increased shortly after birth in both tissues, this concentration being in a newborn animal 0.149 ± 0.027 nmol/mg protein in liver and 0.333 ± 0.082 nmol/mg protein in heart. The respective values from a two week old animal were 0.216±0.031 and 0.583 10.100. Postnatally cytochromes c±c₁ and b increased markedly in the heart, but in the liver of newborn animals the cytochrome content was more close to the adult values. The amount of mitochondrial protein increased after birth, too. In a newborn animal the mitochondrial protein values were 39.7 ± 3.6 mg/g wet weight in liver and 26.6± 6.5 mg/g wet weight in heart. In adult animals the respective values were 71.5 ± 4.8 and 80.7± 13.1. The effect of postnatal hypoxia (10% O₂, 24 h and 48 h) on liver and heart mitochondrial cytochrome concentration and protein values of newborn animals were investigated. During hypoxia the amount of mitochondrial protein remained about at the level of a newborn animal. The postnatal increase in the mitochondrial cytochrome concentration, which was smaller in the liver than in the heart, was also inhibited by hypoxia.     

Redistribution of Cytochrome c Precedes the Caspase-Dependent Formation of Ultracondensed Mitochondria, with a Reduced Inner Membrane Potential, in Apoptotic Monocytes

Apoptosis was induced in human monocytic THP.1 cells by the use of chemicals with disparate mechanisms of action. Apoptotic cells were characterized by a reduced inner mitochondrial membrane potential, increased cytosolic cytochrome c, ultracondensed mitochondria, condensed chromatin, cytoplasmic inclusions of beta-actin, and fragmentation of the Golgi apparatus. All of these changes, except the release of cytochrome c, were prevented by caspase inhibition. Cells were separated into two populations, with either normal or low inner mitochondrial membrane potential, using fluorescence-activated cell sorting. Ultracondensed mitochondria were observed only in the cells with low inner mitochondrial membrane potential, whereas noncondensed mitochondria were found in the cells with a normal inner mitochondrial membrane potential. We have demonstrated a sequence of related biochemical and ultrastructural changes, commencing with the release of mitochondrial cytochrome c, followed by activation of caspases and a reduction of inner mitochondrial membrane potential. These changes involved the formation of ultracondensed but not swollen mitochondria. Thus the release of mitochondrial cytochrome c was not the result of the mitochondrial permeability transition, reduction of inner mitochondrial membrane potential, or rupture of the outer mitochondrial membrane. Discontinuities in the outer membrane of ultracondensed mitochondria may, however, facilitate the further release of caspase-activating proteins, thereby amplifying the apoptotic process.     

Age-dependent changes in pyruvate uptake by nonsynaptic and synaptic mitochondria from rat brain

Changes in the uptake of pyruvate by nonsynaptic and synaptic mitochondria from brains of young adult and old rats were investigated. An age-dependent decrease in State 3 respiration in the presence of pyruvate plus malate as substrate was observed in cerebral mitochondrial populations but not in liver mitochondria. Addition of exogenous cytochrome c to nonsynaptic and synaptic mitochondria enhanced the rate of State 3 respiration but the age-dependent decrease in State 3 respiration persisted in both types of mitochondria. A decrease in the uptake of pyruvate as measured by the inhibitor-stop and rapid centrifugation techniques was observed in both nonsynaptic and synaptic mitochondria from 24-month-old rats compared to 3-month-old rats. The results suggest that the decrease in the uptake of pyruvate may be one of the factors responsible for the observed reduction in State 3 respiration in the presence of pyruvate plus malate by both nonsynaptic and synaptic mitochondria from brains of senescent rats compared to young adults.     

严重烧伤早期心肌[Ca2+]m变化及其机制研究

目的:探讨严重烧伤早期心肌线粒体Ca²⁺浓度([Ca²⁺]_m)的动态变化规律及其发生机制。方法:复制30%Ⅲ°烫伤大鼠模型,测定伤后1、3、6、12、24h大鼠心肌[Ca²⁺]_m,同时检测影响[Ca²⁺]_m的相关指标—胞浆Ca^2＋浓度(_c)及线粒体Ca^2＋转运速率。结果:烧伤后1、3、6h[Ca²⁺]_m依次升高,12、24h较6h虽有所下降,但仍高于正常对照组;_c除伤后1h无明显变化外,其余各时相点变化趋势与[Ca²⁺]_m相同,且伤后[Ca²⁺]_m与_c呈显著正相关,相关系数为0.9177(P＜0.01)。伤后1h心肌线粒体Ca^2＋摄取速率明显升高,而Ca^2＋释放速率无明显改变,但3、6、12、24h心肌线粒体Ca^2＋摄取速率与Ca^2＋释放速率均显著降低,且烧伤后3、6、12、24h[Ca²⁺]_m分别与线粒体Ca^2＋释放速率呈明显负相关。结论:烧伤后心肌线粒体存在明显的Ca^2＋超载和转运紊乱。     

In-vitro translation of mitochondrial mRNAs by yeast mitochondrial ribosomes is hampered by the lack of start-codon recognition

Summary In an attempt to reconstitute an homologous in-vitro translation system for yeast mitochondrial mRNAs, we have isolated ribosomes, supernatant factors, and tRNAs from mitochondria of Saccharomyces carlsbergensis. While poly(U) is translated faithfully in this system, no translation of in-vitro synthesised cytochrome c oxidase subunit II (COX2) mRNA could be detected. Formation of formylmethionyl-puromycin on mitochondrial ribosomes is stimulated by ApUpG, but not by COX2 mRNA, although mitochondrial small ribosomal subunits bind to this mRNA in vitro, even without added tRNA and initiation factors. We conclude, therefore, that the inability to faithfully translate mitochondrial mRNAs in vitro may be the result of an inability of mitochondrial ribosomes to recognize the initiation codon.     

Bacterial LPS Mediated Acute Inflammation-induced Spermatogenic Failure in Rats: Role of Stress Response Proteins and Mitochondrial Dysfunction

Bacterial Lipopolysaccharide (LPS) induced inflammation is implicated in the infection associated testicular tissue damage. Earlier, using a LPS induced acute endotoxemic rat model, we have shown the involvement of inflammation-induced oxidative stress in the impaired steroidogenesis and spermatogenesis. In the present study, we report a significant induction (more than 2-fold) of stress response proteins HSP-60, HMGB-1 and 2 in the testes, as early as 6 h after LPS injection with a later decrease. This induction of acute stress is closely followed by a significant reduction (74%) in Bcl2/Bax ratio along with leakage of cytochrome c (3 fold increase, p < 0.05) from mitochondria and increased caspase-3 activity levels (2.9 fold, p < 0.05) at 12 h and 24 h post LPS injection respectively. Further studies on PARP cleavage revealed a pattern similar to necrotic death during early periods (3 h to 24 h) and apoptosis at later periods (24 h to 72 h) after LPS treatment. In conclusion, the present study shows the involvement of stress response proteins and mitochondrial dysfunction in LPS-induced germ cell death in male rats.     

Effects of hypoxia and fasting on the cytochrome concentration in intestinal epithelial villous cell mitochondria: Role of changes in the life-span of the cells

The effects of hypoxia in vivo (40.8 kPa barometric pressure up to 120h) and fasting on the characteristics of intestinal epithelial villous cell mitochondria and the turnover of epithelial villous cells and mitochondria were studied in rats. Using cells and mitochondria isolated in the isotonic mannitol medium, it was found that 24-h hypoxia or fasting did not alter the mitochondrial cytochrome content, but 48-h hypoxia or fasting led to increases of 70% and 37% in the cytochrome aa₃ concentration in the hypoxic and fasting animals respectively. The turnover of intestinal epithelial cells was studied by observing the labelling kinetics of the cells with ³H-thymidine and the turnover of the cell and mitochondrial proteins with (guanido-¹⁴C)-arginine or ³H-leucine. The decay in thymidine radioactivity obeyed exponential kinetics from which half-lives of 1.15, 1.31 and 1.53 days were calculated in the control, fasting and hypoxic animals respectively. The half-lives for total cellular protein were 1.31, 1.54 and 1.54 days respectively when calculated from the (guanido-¹⁴C)-arginine experiments, or 0.69, 0.75 and 0.99 days when calculated from the leucine experiments. The labelling experiments with (guanido-¹⁴C)-arginine indicated that the turnover of mitochondrial proteins in intestinal epithelial cells is the same as that of the cells themselves. Since the turnover of mitochondrial proteins in other tissues is known to be a relatively slow process, the increase in the cytochrome concentration in the intestinal cells of the hypoxic rats must be due to the longer life of the cells, which allows for the synthesis of larger amounts of the mitochondrial components.     

Mss51p, a putative translational activator of cytochrome c oxidase subunit-1 (COX1) mRNA, is required for synthesis of Cox1p in Saccharomyces cerevisiae

 Mutants of Saccharomyces cerevisiae that lack a functional MSS51 gene are respiratory deficient due to the absence of cytochrome c oxidase subunit 1 (Cox1p). It has been previously suggested, but not formally proven, that Mss51p is required for translational activation of COX1 mRNA, rather than being involved in a subsequent step in the synthesis of Cox1p or its assembly into cytochrome c oxidase. Pulse-chase labelling experiments now show that the absence of detectable levels of Cox1p in mss51-null strains is indeed due to the lack of synthesis of Cox1p, and is not caused by reduced stability of the protein. To gain more insight into the exact function of Mss51p, we determined the subcellular localization of the protein. We were able to show that an epitope-tagged version of Mss51p (Mss51HA) complements the mutation and can be localized in mitochondria, where it is firmly associated with the mitochondrial inner membrane. In addition, we characterized the previously identified mutant allele mss51-3. Sequence analysis revealed the presence of a short open reading frame upstream of MSS51 resulting from the creation of an extra ATG startcodon. Received: 3 December 1999 / 5 January 2000     

Distribution of ryanodine receptors in rat ventricular myocytes

Ryanodine receptors (RyRs) are the major ion channels in the sarcoplasmic reticulum responsible for Ca²⁺ release in muscle cells. Localization of RyRs is therefore critical to our understanding of Ca²⁺ cycling and Ca²⁺-dependent processes within ventricular cells. Recently, RyRs were reportedly found in non-classical locations in the middle of the sarcomere, between perinuclear mitochondria and in the inner mitochondrial membrane of cardiac mitochondria. However, for multiple reasons these reports could not be considered conclusive. Therefore, we modified immunogold labeling to visualize the distribution of RyRs in ventricular myocytes. Using antibodies to the voltage-dependent anion channel (i.e. VDAC) or cytochrome c along with our labeling method, we showed that these mitochondrial proteins were appropriately localized to the mitochondrial outer and inner membrane respectively. Immunogold labeling of ultrathin sections of intact and permeabilized ventricular myocytes with antibodies to three types of RyRs confirmed the existence of RyRs between the Z-lines and around the perinuclear mitochondria. However, we did not find any evidence to support localization of RyRs to the mitochondrial inner membrane.     

Oxa1p, which is required for cytochrome c oxidase and ATP synthase complex formation, is embedded in the mitochondrial inner membrane

We have previously isolated the yeast nuclear gene OXA1 and showed that Oxa1p is required for the formation of the cytochrome c oxidase and ATP synthase complexes. We have expressed Oxa1p in E. coli and shown that it is toxic and rapidly degraded. Nevertheless, a truncated protein was successfully expressed and antibodies have been raised against this truncated protein. These antibodies recognise a protein in mitochondrially enriched fractions. In vitro mitochondrial import experiments demonstrate that the import of Oxa1p is accompanied by the cleavage of a long pre-sequence. Osmotic swelling and alkaline carbonate extraction show that Oxa1p is an integral membrane protein located in the inner membrane of mitochondria. The relationships between the sub-mitochondrial location and the function of Oxa1p are discussed. Received: 9 December 1996 / 21 January 1997