Diana S. Beattie

Studies on the induction of hepatic δ-aminolevulinic acid synthetase in rat liver mitochondria

Abstract Administration of allylisopropylacetamide (AIA) to rats caused an initial induction of hepatic δ-aminolevulinic acid (ALA) synthetase activity in the postmitochondrial supernatant fraction of the cell. The activity in this fraction decreased 50% from 1–3 hr after injection, while during this time period, the ALA synthetase activity of the mitochondrial fraction continued to increase at a nearly linear rate. Cycloheximide blocked the induction of ALA synthetase in both subcellular fractions. Two injections of chloramphenicol inhibited 50–60% the induction of ALA synthetase activity in the mitochondrial fraction but not in the postmitochondrial supernatant fraction. The apparent half-life of the enzyme was 66 min after cycloheximide administration and 118 min after chloramphenicol administration. A slower rate of turnover of the enzyme was observed in animals 1 hr after AIA injection. These results support the suggestion that ALA synthetase is synthesized in the microsomes and is transferred to the mitochondrial matrix in a subsequent step. The content of cytochromes a–a3, b, c–c1 was increased 50% in mitochondria which contained high ALA synthetase activity.

The effect of diabetes on protein synthesis and the respiratory chain of rat skeletal muscle and kidney mitochondria.

Abstract The effects of streptozotocin-induced diabetes mellitus upon mitochondria from rat skeletal muscle and kidney were examined. The rate of amino acid incorporation in vitro by isolated skeletal muscle mitochondria from diabetic animals was decreased by 50–60% from control values. Treatment of diabetic animals with insulin lowered blood glucose levels to control values and restored the rate of muscle mitochondrial protein synthesis in vitro to control levels. The rates of skeletal muscle mitochondrial protein synthesis were also decreased 23–27% by a 2-day fast. Comparison of the translation products synthesized by isolated muscle mitochondria from control and diabetic rats by dodecyl sulfate polyacrylamide-gel electrophoresis revealed a uniform decrease in the synthesis of all polypeptides. Aurintricarboxylic acid and pactamycin, inhibitors of chain initiation, blocked protein synthesis to a greater extent in muscle mitochondria from control as compared to diabetic animals suggesting that mitochondria from diabetics are unable to initiate protein synthesis at a rate comparable to control. Phenotypic changes observed in diabetic muscle mitochondria included a 36% decrease in the content of cytochromes aa 3 and a 27% decrease in cytochrome b , both established as containing mitochondrial translation products in lower eucaryotes. State 3 respiration with glutamate as substrate decreased by 27% and uncoupler-stimulated respiration decreased by 23% in the diabetic mitochondria. By contrast, the specific activities of NADH and succinate dehydrogenases, established as products of cytoplasmic protein synthesis in lower eucaryotes, were not decreased in skeletal muscle mitochondria from the diabetic animals. These results suggest that the considerable muscular atrophy observed in diabetics may involve decreases in both cytoplasmic and mitochondrial protein synthesis, the latter reflected in profound changes in the respiratory chain. By contrast, comparison of kidney mitochondria from control and diabetic rats revealed no differences in the rates of protein synthesis in vitro , nor in the mitochondrial translation products, which corresponded closely to liver and skeletal muscle translation products. Similarly, the mitochondrial content of cytochromes b , c + c 1 , and aa 3 , the specific activity of succinate dehydrogenase, the rate of state 3 respiration, and the recovery of mitochondria from kidney homogenates did not differ in control and diabetic animals. Kidney mitochondria are thus like liver mitochondria in being relatively unaffected by insulin deprivation.

Mitochondrial protein synthesis in vitro is not an artifact

The first report that mitochondria, in vitro, can incorporate amino acids into protein was published in 1958 [ 11. Since that time, numerous studies have detailed this process in mitochondria isolated from mammalian and insect tissues, as well as from cultured mammalian cells, yeast, fungi, and plants (cf. [2, 31). In these studies, it was demonstrated that amino acid incorporation by isolated mitochondria was not due to either microsomal [4] or bacterial contamination [5, 61. In recent publications, Hochberg et al. [7, 81 have suggested that mitochondrial protein synthesis in vitro may be an artifact resulting from specific binding of radioactive amino acids to mitochondrial protein-lipid structures. We have carefully reinvestigated this problem and observed that three different concentrations of leucine are not incorporated into heat or acid-denatured mitochondria significantly above the zero time values. Under the conditions used in our laboratory, leucine is incorporated into proteins of intact mitochondria at a rate nearly 20-fold greater than that reported by Hochberg et al. [7]. Possible reasons for these differences will be discussed as well as other evidence which demonstrates that protein synthesis by isolated mitochondria in vitro is not an artifact.

δ-Aminolevulinic acid synthetase: Regulation of activity in various tissues of the aging rat

Abstract The basal and ethanol-induced activities of the rate-limiting enzyme of heme biosynthesis, δ-aminolevulinic acid (ALA) synthetase were measured in the liver, heart, kidney, and brain of young, adult, and aged Sprague-Dawley rats. When assayed in whole mitochondria derived from either fed or 24-h fasted animals, the basal levels of hepatic ALA synthetase activity decreased dramatically as a function of age. An equivalent decrease was seen in the ethanol-induced activity although the ratio of induced to basal activities did not change with age. In the heart, ALA synthetase activity also decreased significantly during aging. The activity was not induced by ethanol and was decreased markedly by fasting. By contrast, kidney ALA synthetase activity showed no age-related changes. The activity was unaffected by fasting and showed a variable induction response to ethanol. Brain ALA synthetase activity displayed a significant age-dependent decrease in its activity which was neither affected by fasting nor sensitive to induction by ethanol. The data presented are consistent with the hypothesis that ALA synthetase activity is subject to metabolic regulation. Further, they indicate that while the enzyme activity is regulated in a tissuespecific manner, a time-dependent decrease is a general feature of the aging animal.

The relationship of protein and lipid synthesis during the biogenesis of mitochondrial membranes

SummaryRat liver mitochondria were fractionated into inner and outer membrane components at various times after the intravenous injection of14C-leucine or14C-glycerol. The time curves of protein and lecithin labeling were similar in the intact mitochondria, the outer membrane fraction, and the inner membrane fraction. In rat liver slices also, the kinetics of3H-phenylalanine incorporation into mitochondrial KCl-insoluble proteins was identical to that of14C-glycerol incorporation into mitochondrial lecithin. These results suggest a simultaneous assembly of protein and lecithin during membrane biogenesisThe proteins and lecithin of the outer membrane were maximally labeledin vivo within 5 min after injection of the radioactive precursors, whereas the insoluble proteins and lecithin of the inner membrane reached a maximum specific acitivity 10 min after injection.Phospholipid incorporation into mitochondria of rat liver slices was not affected when protein synthesis was blocked by cycloheximide, puromycin, or actinomycin D. The injection of cycloheximide 3 to 30 min prior to14C-choline did not affect thein vivo incorporation of lecithin into the mitochondrial inner or outer membranes; however treatment with the drug for 60 min prior to14C-choline resulted in a decrease in lecithin labeling. These results suggest that phospholipid incorporation into membranes may be regulated by the amount of newly synthesized protein available.When mitochondria and microsomes containing labeled phospholipids were incubated with the opposite unlabeled fractionin vitro, a rapid exchange of phospholipid between the microsomes and the outer membrane occurred. A slight exchange with the inner membrane was observed.

Formation of the yeast mitochondrial membrane: III. Accumulation of mitochondrial proteins synthesized in both the cytoplasm and mitochondria in yeast undergoing glucose derepression

Abstract The inhibitors of protein synthesis, chloramphenicol and cycloheximide, were added to cultures of yeast undergoing glucose derepression at different times during the growth cycle. Both inhibitors blocked the increase in activity of coenzyme QH 2 -cytochrome c reductase, suggesting that the formation of complex III of the respiratory chain requires products of both mitochondrial and cytoplasmic protein synthesis. The possibility that precursor proteins synthesized by either cytoplasmic or mitochondrial ribosomes may accumulate was investigated by the sequential addition of cycloheximide and chloramphenicol (or the reverse order) to cultures of yeast undergoing glucose derepression. When yeast cells were grown for 3 hr in medium containing cycloheximide and then transferred to medium containing chloramphenicol, the activity of cytochrome oxidase increased at the same rate as the control during the first hour in chloramphenicol. These results suggest that some accumulation of precursor proteins synthesized in the mitochondria had occurred when cytoplasmic protein synthesis was blocked during the growth phase in cycloheximide. In contrast, essentially no products of mitochondrial protein synthesis accumulated as precursors for either oligomycin-sensitive ATPase or complex III of the respiratory chain during growth of the cells in cycloheximide. When yeast were grown for 3 hr in medium containing chloramphenicol followed by 1 hr in cycloheximide, the activities of cytochrome oxidase and succinate-cytochrome c reductase increased at the same rate as the control, while the activities of oligomycin-sensitive ATPase and NADH or coenzyme QH 2 -cytochrome c reductase were nearly double that of the control. These data suggest that a significant accumulation of mitochondrial proteins synthesized in the cytoplasm had occurred when the yeast cells were grown in medium containing sufficient chloramphenicol to block mitochondrial protein synthesis. The possibility that proteins synthesized in the cytoplasm may act to control the synthesis of mitochondrial proteins for both oligomycin-sensitive ATPase and complex III of the respiratory chain is discussed.

Products of rat liver mitochondrial protein synthesis: Electrophoretic analysis of the number and size of these proteins and their solubility in chloroform: Methanol

Abstract Rat liver mitochondria were incubated in vitro with radioactive leucine, and submitochondrial particles prepared by several methods. Analysis of the labeled mitochondrial membrane fractions by sodium dodecylsulfate gel electrophoresis revealed three labeled bands of molecular weights corresponding to 40,000; 27,000; and 20,000 daltons. Electrophoresis for longer times at higher concentrations of acrylamide revealed eight labeled bands, ranging in molecular weights from 48,000 to 12,000. Mitochondria were incubated for 5 min with [ 3 H]leucine followed by a chase of unlabeled leucine. Gel electrophoresis of the membranes obtained after labeling for 5 min indicated significant synthesis of polypeptides in the 40,000 M r , range and very little labeling of low molecular-weight polypeptides. After addition of the chase, increased synthesis of the high molecular-weight polypeptides was observed; however, no significant increase or decrease of radioactivity in the bands of low molecular-weight was observed, suggesting that rat liver mitochondria have the ability to synthesize complete proteins in the M r 27,000–40,000 range. Approximately 16% of the total leucine incorporated into protein by isolated rat liver mitochondria in vitro could be extracted by chloroform: methanol. Gel electrophoresis of the chloroform: methanol extract revealed several bands containing radioactivity with the majority of counts in a band of 40,000 molecular weight. Gel electrophoresis of the chloroform: methanol extract of lyophilized submitochondrial particles indicated label in two broad bands in the low molecular-weight region of 14,000-10,000 with insignificant counts in the higher molecular-weight regions of the gel. Yeast cells were pulse labeled in vivo with [ 3 H]leucine in the presence of cycloheximide and the submitochondrial particles extracted with chloroform:methanol. The extract separated after gel electrophoresis into four labeled bands ranging in molecular weight from 52,000 to 10,000. Preincubation of the yeast cells with chloramphenicol prior to the pulse labeling caused a 6-fold stimulation of labeling into the band of lowest molecular weight of the chloroform: methanol extract. These results suggest that the accumulation of mitochondrial proteins synthesized in the cytoplasm, when chloramphenicol is present in the medium, may stimulate the synthesis of certain specific mitochondrial proteins which are soluble in chloroform: methanol.

Decreased amounts of core proteins I and II and the iron-sulfur protein in mitochondria from yeast lacking cytochrome b but containing cytochrome c1.

The effect of cytochrome b on the assembly of the subunits of complex III into the inner mitochondrial membrane has been studied in four mutants of yeast that lack a spectrally detectable cytochrome b and do not synthesize apocytochrome b. Quantitative analysis of intact mitochondria by immunoprecipitation or immunoblotting techniques with specific antisera revealed that the core proteins and the iron-sulfur protein were decreased 50% or more in the mitochondria from the mutants as compared to the wild type. Sonication of wild-type mitochondria did not result in any decrease in any of these proteins from the membrane; however, sonication of mitochondria from the four mutants resulted in a further decrease in the amount of these proteins suggesting that they are not as tightly bound to the mitochondrial membrane in the absence of cytochrome b. By contrast, the amounts of cytochrome c1 in the mitochondria, as determined both spectroscopically and immunologically, were not significantly affected by the absence of cytochrome b. In addition, no loss of cytochrome c1 was observed after sonication of the mitochondria suggesting that this protein is tightly bound to the membrane. These results suggest that the processing and/or assembly of these subunits of complex III into the mitochondrial membrane is affected by the absence of cytochrome b.

The possible relationship between heme synthesis and mitochondrial biogenesis.

Abstract Administration of allylisopropylacetamide (AIA) 1 to rats causes a rapid induction of hepatic δ-aminolevulinic acid (ALA) synthetase. Liver mitochondria obtained from animals after AIA treatment had a 50–60% greater rate of amino acid incorporation in vitro as compared to mitochondria from control animals. Similarly, pulselabeling by ( 3 H) leucine in vivo was stimulated 50% in submitochondrial fractions containing proteins insoluble in acetic acid and a membranous preparation of cytochrome oxidase. Previous studies had indicated that 15–20% of the proteins in these fractions were labeled in vivo by a cycloheximide-insensitive system, presumably that of the mitochondria ( Fed. Eur. Biol. Soc. Lett. 9, 232 1970). In time-course studies, it was observed that increased incorporation of ( 14 C)glycine into heme in vivo followed shortly after ALA synthetase induction. A slight decay occurred before the incorporation of ( 3 H)leucine into cytochrome oxidase or total mitochondrial protein was stimulated. At these latter times a 20–30% increase in the cytochrome content was also observed. Administration of aminotriazole, an inhibitor of ALA dehydratase and hence overall heme synthesis, prevented the stimulation of leucine incorporation in vivo and the increase in cytochrome content observed after AIA treatment. These results suggest that mitochondrial protein synthesis and cytochrome formation may be stimulated when heme biosynthesis is increased due to ALA synthetase induction.

Orientation of complex III in the yeast mitochondrial membrane: Labeling with [125I] diazobenzenesulfonate and functional studies with the decyl analogue of coenzyme Q as substrate

Mitochondria (or mitoplasts) and submitochondrial particles from yeast were treated with [125I] diazobenzenesulfonate to label selectively proteins exposed on the outer or inner surface of the inner mitochondrial membrane. Polyacrylamide gel analysis of the immunoprecipitates formed with antibodies against Complex III or cytochromeb revealed that the two core proteins and cytochromeb were labeled in both mitochondria and submitochondrial particles, suggesting that these proteins span the membrane. Cytochromec1 and the iron sulfur protein were labeled in mitochondria but not in submitochondrial particles, suggesting that these proteins are exposed on the cytosolic side of the inner membrane. The steady-state reduction of cytochromesb andc1 was determined with succinate and the decyl analogue of coenzyme Q as substrates. Addition of the coenzyme Q analogue to mitochondria caused reduction of 15–30% of the total dithionite-reducibleb and 100% of the cytochromec1: Addition of the coenzyme Q analogue to submitochondrial particles led to the reduction of 70% of the total dithionite-reducible cytochromeb but insignificant amounts of cytochromec1. A model to explain the topography of Complex III in the inner membrane is proposed based on these results.