Regulation of mitochondrial membrane protein turnover by thyroid hormone(s)

Effects of thyroidectomy on turnover rate of proteins of rat liver mitochondria, mitochondrial membranes and microsomes were examined. Thyroidectomy resulted in a significant increase in the half-lives of whole mitochondria and inner membrane-matrix, the effect being less pronounced on the half-lives of outer mitochondrial membrane and microsomes.     

Partial purification and characterization of citrate synthase from Dictyostelium discoideum.

The effect of thyroidectomy on oxidative metabolism of rat liver, kidney, and brain mitochondria has been examined. The respiration in liver, kidney, and brain mitochondria was affected differentially after thyroidectomy, the common effect in all the tissues being the impairment in state 3 as well as state 4 rates of succinate oxidation. Thyroidectomy did not have any effect on $\text{ADP}\text{O}$ ratios; however, compared to normal, respiratory control indexes were, in general, somewhat higher. Thyroidectomy also did not alter total ATPase activity of liver, kidney, and brain mitochondria, although the basal ATPase activity had decreased significantly under these conditions. The cytochrome content of the mitochondria also showed tissue-specific changes after thyroidectomy; however, no significant changes in the absorption characteristics of the cytochromes were seen. The succinate and glutamate dehydrogenase activities of mitochondria from liver, kidney, and brain were not affected by thyroidectomy, thereby ruling out the possibility that the decrease in substrate oxidation may be due to alterations in the primary dehydrogenase levels. It is concluded that thyroid hormone(s) may have a tissue-specific role in regulating the metabolic functions of mitochondria.     

Influence of thyroid status on the electrophoretic pattern of rat liver mitochondrial proteins]

Liver mitochondria were isolated from normal and thyroidectomized rats and their protein components analyzed by polyacrylamide gel electrophoresis. In whole mitochondria 35 protein fractions with MW ranging from 10,000 to 135,000 were characterized. In the absence of thyroid hormone secretion, the amount of a MW 54,000 fraction was always decreased. Injection of small doses of 3,5,3'-triiodo-L-thyronine to the thyroidectomized animal restored the quantity of that protein fraction to normal. Isolated outer mitochondrial membranes showed the presence of 20 protein fractions. These fractions revealed no change after thyroidectomy. The mitoplast, which contained 35 fractions, exhibited a decrease of the MW 54,000 component in thyroidectomized rats. The mitoplast was separated into several fractions. Water soluble matrix proteins presented molecular weights ranging between 40,000 and 55,000. Proteins, which were slightly bound to the inner mitochondrial membrane and could be extracted by KCl, presented molecular weights between 25,000 and 45,000. Structural proteins showed a principal specific component of MW equals 23,000. Electrophoretic patterns obtained with these submitochondrial fractions were similar in normal and thyroidectomized animals. The mitoplast fraction which contained the insoluble cytochromes (a, a3, b, c1) was isolated ; its principal constituent, of MW 54,000 was significantly decreased after thyroidectomy. Thus, the lack of thyroid hormone secretion lowered the level of a protein constituent bound to the inner membrane of liver mitochondria. The synthesis of this constituent could be controlled by mitochondrial nucleic acids.     

Transport of proteins into hepatic and nonhepatic mitochondria: specificity of uptake and processing of precursor forms of carbamoyl-phosphate synthetase I

An in vitro system reconstituted with mouse liver polysome translation products was used to study the nature of polypeptide species imported into mitochondria from different mouse tissues such as liver, kidney, brain, and heart, as well as from Ehrlich ascites, Novikoff hepatoma, and Morris hepatoma 3924A tumor lines. Mouse hepatic mitochondria import a number of proteins including 160-kilodalton (kDa) carbamoyl-phosphate synthetase I (CPS-I). Two other proteins of 63 and 57 kDa of unknown function are also imported as major components by mouse liver mitochondria. Under these in vitro conditions, however, mitochondria from non-CPS-I expressing tissues such as brain, kidney, and heart failed to import and process the precursor forms of CPS-I (pCPS-I). Furthermore, mitochondria from three different tumor lines (Novikoff hepatoma, Morris hepatoma, and Ehrlich ascites) containing negligible CPS-I activity were also unable to import and process pCPS-I to any significant level. Similarly, the 63-kDa protein was selectively transported into liver and kidney mitochondria and also into Ehrlich ascites mitochondria at reduced levels, but not into mitochondria from heart and brain. Nevertheless, the 57-kDa protein and a number of proteins of less than 45 kDa are transported efficiently by all of the mitochondrial types studied. These results provide evidence for tissue- or cell-specific selectivity at the mitochondrial membrane level for the transport of some proteins. The transports of 63- and 57-kDa proteins are differentially inhibited by mouse liver mitochondrial matrix and membrane fractions, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biogenesis of mammalian mitochondria in the renoprival kidney. II. Aspects of mitochondrial DNA synthesis

Mitochondrial DNA (m-DNA) content and factors which might control its concentration were investigated in the renoprival kidney at various times after unilateral nephrectomy. On the basis of mitochondrial protein, m-DNA increased 30% in the renoprival kidney at 24 hr and returned to normal by 48 hr. The total tissue content of m-DNA was also increased at 24 hr. The specific activity of [³H]thymidine incorporated into m-DNA in vivo was decreased markedly at 24 hr after mononephrectomy; at the same time there was a threefold increase of [³H]thymidine incorporation into total cellular DNA. The incorporation into m-DNA was above normal at 48 hr. The mitochondrial specific DNase was decreased 60% at 24 and 36 hr post-mononephrectomy. There was no significant difference in the total radioactivity or total optical density at 260 nm of the acid soluble extract from mitochondria isolated at various times after mononephrectomy. The incorporation of [³H]thymidine into TMP and TDP in the renoprival kidney was not different from normal but there was a decrease in the incorporation into TTP. It is suggested that the increase in mitochondrial DNA could be due to a decrease in the rate of degradation rather than an increase in synthesis.     

Identification of mitochondrial branched chain aminotransferase and its isoforms in rat tissues.

The tissue distribution and subcellular location of branched chain aminotransferase was analyzed using polyclonal antibodies against the enzyme purified from rat heart mitochondria (BCATm). Immunoreactive proteins were visualized by immunoblotting. The antiserum recognized a 41-kDa protein in the 100,000 x g supernatant from a rat heart mitochondrial sonicate. The 41-kDa protein was always present in mitochondria which contained branched chain aminotransferase activity, skeletal muscle, kidney, stomach, and brain, but not in cytosolic fractions. In liver mitochondria, which have very low levels of branched chain aminotransferase activity, the 41-kDa protein was not present. However, two immunoreactive proteins of slightly higher molecular masses were identified. These proteins were located in hepatocytes. The 41-kDa protein was present in fetal liver mitochondria but not in liver mitochondria from 5-day neonates. Thus disappearance of the 41-kDa protein coincided with the developmental decline in liver branched chain aminotransferase activity. Two-dimensional immunoblots of isolated BCATm immunocomplexes showed that the liver immunoreactive proteins were clearly different from the heart and kidney proteins which exhibited identical immunoblots. Investigation of BCATm in subcellular fractions prepared from different skeletal muscle fiber types revealed that branched chain aminotransferase is exclusively a mitochondrial enzyme in skeletal muscles. Although total detergent-extractable branched chain aminotransferase activity was largely independent of fiber type, branched chain aminotransferase activity and BCATm protein concentration were highest in mitochondria prepared from white gastrocnemius followed by mixed skeletal muscles with lowest activity and protein concentration found in soleus mitochondria. These quantitative differences in mitochondrial branched chain aminotransferase activity and enzyme protein content suggest there may be differential expression of BCATm in different muscle fiber types.     

Effect of administration of diethylhexyl phthalate on the function and turnover of rat hepatic mitochondria

Administration of the widely used plasticizer di(2-ethylhexyl)phthalate (2% w/w) in the diet to the rat caused proliferation of mitochondria in the liver. The number of mitochondria as well as the amount of protein recovered in the organellar fraction was doubled. Mitochondria isolated from the livers of treated animals showed decreased (50%) respiratory activity. The content and activity of cytochrome oxidase were also decreased. The specific incorporation of amino acids into the proteins of whole liver and of mitochondria was not increased in plasticizer-treated animals. Isolated mitochondria also did not show any difference in the rate of incorporation of amino acids into proteins. The half-lives of whole liver proteins and of mitochondria were increased in plasticizer-fed animals. The half-life of cytochrome oxidase, however, was unaffected by the treatment. The pattern of double labeling of mitochondrial proteins confirmed decreased turnover in plasticizer-treated animals.     

Alterations in albumin secretion and total protein synthesis in livers of thyroidectomized rats.

Perfused rat livers and isolated rat hepatocytes exhibited a 50% decrease in the secretion of both albumin and total secretory proteins after thyroidectomy. In contrast, synthesis of non-secretory proteins was decreased by only 20% from the rates observed in liver preparations from euthyroid rats. These observations suggested a disproportionate effect of thyroidectomy on the synthesis of secretory proteins compared with non-secretory proteins. Disproportionate decreases in the synthesis of albumin in other endocrine-deficient states such as hypophysectomy and diabetes had previously been shown to be associated with decreases of similar magnitude in the relative abundance of albumin-mRNA sequences. In contrast, thyroidectomy did not affect the activity or amount of albumin mRNA in total liver poly(A)-containing RNA when assayed by cell-free translation and by hybridization with complementary DNA, respectively. Furthermore, labelling experiments in vivo demonstrated that albumin synthesis represented 12.9 +/- 0.5% and 12.4 +/- 0.4% of total protein synthesis in livers of thyroidectomized and euthyroid rats respectively. Therefore the fall in secretion of albumin and total secretory protein after thyroidectomy did not appear to be a reflection of disproportionate decreases in the synthesis of these proteins. Instead, defects in steps involved in the post-synthetic processing and secretion of albumin are suggested. A number of comparisons, including ribosome half-transit times, the size distributions of total and albumin-synthesizing polyribosomes, and the fraction of RNA present as inactive ribosomes, provided evidence that the overall decrease in protein synthesis after thyroidectomy was not due to generalized alterations in translational processes. Instead, the decrease in total protein synthesis appeared to reflect the RNA content of the liver, which fell in proportion to th decrease in protein synthesis.     

Heterogeneity of rat liver mitochondrial fractions and the effect of tri-iodothyronine on their protein turnover

1. Rat liver mitochondria were separated into heavy, light and fluffy fractions by differential centrifugation under standard conditions. 2. All mitochondrial fractions possessed soluble as well as membrane-bound enzymes typical of mitochondria. 3. The heavy fraction represented the stable mitochondrial structures and the fluffy particles appear to be loosely coupled. 4. The light mitochondrial fraction lacked the ability of coupled phosphorylation. 5. A study of mobility and isoelectric pH indicated a similarity in the basic membrane structure of all the mitochondrial fractions. 6. The turnover rates of proteins in the heavy and fluffy particles were almost identical; however, this rate was rapid for the light mitochondrial fraction. 7. On treatment with 3,3',5-tri-iodo-l-thyronine, succinoxidase activity was maximally stimulated much earlier in the light mitochondrial fraction than in the heavy fraction. The activity of the fluffy particles, however, remained almost unaffected. 8. Malate dehydrogenase activity in all the mitochondrial fractions was stimulated only at 40h after tri-iodothyronine treatment. 9. The pattern of incorporation of dl-[1-(14)C]leucine in vivo in the tri-iodothyronine-treated animals indicated a rapid initial incorporation and high synthetic ability of the light mitochondrial fraction. 10. The turnover pattern of proteins of the mitochondrial fractions from animals receiving repeated doses of tri-iodothyronine was remarkably different from the normal pattern and suggested that preformed soluble protein units may be incorporated in the light mitochondrial fraction during maturation to form the stable heavy mitochondria. 11. The amount of light-mitochondrial proteins decreased by 40% on thyroidectomy and increased by 160% on treatment with tri-iodothyronine. 12. The possible significance of these results is discussed in relation to mitochondrial genesis.     

Effects of thyroidectomy of the rat on the structure and functions of skeletal muscle mitochondria]

Mitochondria used in the present study were isolated from skeletal muscle of normal and thyroidectomized rats. The preparations were controlled by electron microscopy. It was not possible to find any morphological change induced by thyroidectomy, nevertheless, some difference appeared in the cytochrome contents which were slightly decreased. Oxygen consumption rates of thyroidectomized rat mitochondria were decreased when the particles were maintained in states 3 and 4 in the presence of various substrates, but the P/O ratios were not modified. The activities of mitochondrial enzymes were in general slightly affected by thyroidectomy except for glycerol-1-phosphate cytochrome c reductase and NADH rotenone sensitive cytochrome c reductase which were decreased and for glutamate dehydrogenase activity which was increased. The tRNA nucleotidyltransferase activity found in the mitochondrial matrix was not influenced by the absence of thyroid secretion. Normal rat muscle mitochondria incorporate 14C-leucine with an artificial ATP-generating system or with a respiratory substrate. The amino acid incorporation was decreased by thyroidectomy. Muscle mitochondria analyzed by polyacrylamide gel electrophoresis contained more than 30 protein components with MW ranging from 10.000 to 135.000. Thyroidectomy lowered the amount of a fraction of about 54.000 MW. It is not impossible that all the data observed in the absence of thyroid secretion are in relation with changes induced in the mitochondrial genome as previously shown in mitochondria isolated from liver or thyroidectomized rats.     

Effect of cisplatin on mitochondrial protein, glutathione, and succinate dehydrogenase in Dalton lymphoma-bearing mice

Cisplatin treatment of tumor-bearing mice resulted a significant decrease of protein in the tissues studied (liver, kidney, and Dalton lymphoma) and also in their mitochondrial fractions. As compared to respective tissues, the protein decrease was noted to be more conspicuous in their mitochondrial fractions. Similarly, mitochondrial glutathione also decreased significantly in the tissues. However, succinate dehydrogenase activity was selectively decreased in the kidney and Dalton lymphoma cells, whereas in liver it remained almost unchanged. An increase in serum urea concentration and kidney mitochondrial lipid peroxidation was also observed after cisplatin treatment. It is suggested that the cisplatin-induced biochemical changes in mitochondria involving mitochondrial protein, glutathione, and succinate dehydrogenase could be the important potent cellular sites contributing to toxicity/cytotoxicity after cisplatin treatment. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of thyroid state on susceptibility to oxidants and swelling of mitochondria from rat tissues

The effects of the thyroid state on oxidative damage, antioxidant capacity, susceptibility to in vitro oxidative stress and Ca(2+)-induced permeabilization of mitochondria from rat tissues (liver, heart, and gastrocnemious muscle) were examined. Hypothyroidism was induced by administering methimazole in drinking water for 15 d. Hyperthyroidism was elicited by a 10 d treatment of hypothyroid rats with triiodothyronine (10 micro g/100 g body weight). Mitochondrial levels of hydroperoxides and protein-bound carbonyls significantly decreased in hypothyroid tissues and were reported above euthroid values in hypothyroid rats after T(3) treatment. Mitochondrial vitamin E levels were not affected by changes of animal thyroid state. Mitochondrial Coenzyme Q9 levels decreased in liver and heart from hypothyroid rats and increased in all hyperthyroid tissues, while Coenzyme Q10 levels decreased in hypothyroid liver and increased in all hyperthyroid tissues. The antioxidant capacity of mitochondria was not significantly different in hypothyroid and euthyroid tissues, whereas it decreased in the hyperthyroid ones. Susceptibility to in vitro oxidative challenge decreased in mitochondria from hypothyroid tissues and increased in mitochondria from hyperthyroid tissues, while susceptibility to Ca(2+)-induced swelling decreased only in hypothyroid liver mitochondria and increased in mitochondria from all hyperthyroid tissues. The tissue-dependence of the mitochondrial susceptibility to stressful conditions in altered thyroid states can be explained by different thyroid hormone-induced changes in mitochondrial ROS production and relative amounts of mitochondrial hemoproteins and antioxidants. We suggest that susceptibilities to oxidants and Ca(2+)-induced swelling may have important implications for the thyroid hormone regulation of the turnover of proteins and whole mitochondria, respectively.     

A calmodulin endoproteinase from mitochondrial membranes

A proteinase specific for calmodulin has been identified in a crude rat kidney Triton-extracted or sonicated mitochondrial fraction and solubilized by EGTA extraction of these membranes. Mitochondrial fractions from other tissues had less activity, with relative activities: kidney = spleen greater than testes greater than liver, and no detectable activity in either brain or skeletal muscle. This enzyme is active in the presence of EGTA, but not in the presence of calcium, and cleaves calmodulin into three major peptide fragments with Mr 6000, 9000 and 10,000. N-methylated and non-methylated calmodulins were both cleaved by calmodulin proteinase and while troponin was a poor substrate, it was cleaved in the presence of either calcium or EGTA. No other EF hand calcium-binding proteins or other major mitochondrial proteins were cleaved by this enzyme. The peptides resulting from calmodulin proteinase action were isolated by reverse-phase high performance liquid chromatography (HPLC) and sequenced. Sequence analysis indicated that calmodulin proteinase cleaves calmodulin at Lys-75. The effects of proteinase inhibitors indicate that calmodulin proteinase is a trypsin-like enzyme belonging to the serine endopeptidase family of enzymes.     

The mitochondrial energy transduction system and the aging process

Aged mammalian tissues show a decreased capacity to produce ATP by oxidative phosphorylation due to dysfunctional mitochondria. The mitochondrial content of rat brain and liver is not reduced in aging and the impairment of mitochondrial function is due to decreased rates of electron transfer by the selectively diminished activities of complexes I and IV. Inner membrane H⁺ impermeability and F₁-ATP synthase activity are only slightly affected by aging. Dysfunctional mitochondria in aged rodents are characterized, besides decreased electron transfer and O₂ uptake, by an increased content of oxidation products of phospholipids, proteins and DNA, a decreased membrane potential, and increased size and fragility. Free radical-mediated oxidations are determining factors of mitochondrial dysfunction and turnover, cell apoptosis, tissue function, and lifespan. Inner membrane enzyme activities, such as those of complexes I and IV and mitochondrial nitric oxide synthase, decrease upon aging and afford aging markers. The activities of these three enzymes in mice brain are linearly correlated with neurological performance, as determined by the tightrope and the T-maze tests. The same enzymatic activities correlated positively with mice survival and negatively with the mitochondrial content of lipid and protein oxidation products. Conditions that increase survival, as vitamin E dietary supplementation, caloric restriction, high spontaneous neurological activity, and moderate physical exercise, ameliorate mitochondrial dysfunction in aged brain and liver. The pleiotropic signaling of mitochondrial H₂O₂ and nitric oxide diffusion to the cytosol seems modified in aged animals and to contribute to the decreased mitochondrial biogenesis in old animals. oxidative damage; survival; complexes I and IV; nitric oxide synthase     

The apparent turnover of mitochondria, ribosomes and sRNA of the brain in young adult and aged rats

—[¹⁴C] orotic acid and [³H]l -leucine were injected intraperitoneally into two groups of rats, aged 12 and 24 months, respectively. The apparent turnover of RNA and protein from several subcellular fractions was assessed by following the loss of label from these fractions with time. The curves for apparent turnover of all protein fractions from mitochondria were single exponential curves. Total mitochondrial protein from younger animals had a half-life of 26.8 days. Two protein subfractions, protein insoluble in cold perchloric acid and chloroform-methanol (residual protein) and protein soluble in chloroform-methanol (C–M protein) had similar half-lives: 26.3 and 26.1 days, respectively. For the older animals the half-lives were 23.5 days for total protein, 17.4 for residual protein and 30.4 for C–M protein. The difference between the two protein subfractions from mitochondria of the older animals suggests an age-associated deviation from the synchrony of synthesis and degration of proteins in this organelle. Further deviation from the unit concept of mitochondrial turnover was seen in the apparent turnover of mitochondrial RNA. Mitochondrial RNA had half-lives of 10.0 and 11.6 days for older and younger animals, respectively, with no significant difference between the groups. No age-associated difference was observed in the apparent turnover of sRNA. This fraction exhibited a double exponential turnover pattern; the first component in both cases had a half-life of about 5–8 days and the second component 13–16 days. Ribosomal RNA and protein from both older and younger animals exhibited multiexponential kinetics but both components, RNA and protein, within each age group appeared to turn over synchronously. Average values for apparent turnover of total ribosomes (RNA and protein) were 18.2 days for the older animals and 7.4 days for the younger animals. The age-associated difference was highly significant P < (0.001).     

Mitochondrial turnover in liver is fast in vivo and is accelerated by dietary restriction: application of a simple dynamic model

'Mitochondrial dysfunction', which may result from an accumulation of damaged mitochondria in cells due to a slowed-down rate of mitochondrial turnover and inadequate removal of damaged mitochondria during aging, has been implicated as both cause and consequence of the aging process and a number of age-related pathologies. Despite growing interest in mitochondrial function during aging, published data on mitochondrial turnover are scarce, and differ from each other by up to one order of magnitude. Here we demonstrate that re-utilization of the radioactively labelled precursor in pulse-chase assays is the most likely cause of significant overestimation of mitochondrial turnover rates. We performed a classic radioactive label pulse-chase experiment using (14)C NaHCO(3), whose (14)C is incorporated into various amino acids, to measure mitochondrial turnover in mouse liver. In this system, the activity of the urea cycle greatly limited arginine dependent label re-utilization, but not that of other amino acids. We used information from tissues that do not have an active urea cycle (brain and muscle) to estimate the extent of label re-utilization with a dynamic mathematical model. We estimated the actual liver mitochondrial half life as only 1.83 days, and this decreased to 1.16 days following 3 months of dietary restriction, supporting the hypothesis that this intervention might promote mitochondrial turnover as a part of its beneficial effects.     

Comparison of turnover rates of proteins of the brain, liver and kidney in mouse in vivo following long term labeling.

Intraperitoneal injection of [14C]tyrosine suspension followed by subcutaneous implantation of a [14C]tyrosine pellet in mice produced a fairly constant specific activity of plasma free tyrosine for 5 days, and for 3-5 days in the tissue free amino acid pool. The specific activity of tyrosine in the tissue (brain, liver, and kidney) free amino acid pool was 75-90% of that in plasma. Incorporation of tyrosine into tissue proteins was followed for 5 days in brain; during this time 33% of tissue proteins were labeled. Incorporation for 68 h in liver and kidney showed labeling of over 70% of the protein of these tissues. These percentages assume a homogeneous tissue free tyrosine pool as the precursor. The rate of incorporation initially was 0.6, 2.8, and 2.0% per h in brain, liver, and kidney protein, respectively. These rates decreased in longer term experiments. The best fit to the incorporation curves was obtained by assuming the following average half-lives for tissue proteins: brain, two compartments, 5.7% with a half-life of 15 h, 94.3% with a half-life of 10 days; liver, a single compartment with a 26-h half-life; kidney, two compartments, 41% with an 18-h half-life, and 59% with a 63-h half-life.     

Effect of thryoidectomy on the properties of rat liver mitochondria during liver regeneration]

The properties of regenerating rat liver mitochondria are not changed by the thyroidectomy. Thus, hepatectomy induces, in thyroidectomized rats as in normal ones, a decrease in the activity of the outer membrane enzymes, a stimulation of oxidative phosphorylations and an increase of the turn over rate of mitochondrial proteins. Thyroid hormones do not seem to have a major effect upon the liver regeneration mechanism.