Phosphate In Mitochondria Research Articles

Co-precipitation of Phosphate and Iron Limits Mitochondrial Phosphate Availability in Saccharomyces cerevisiae Lacking the Yeast Frataxin Homologue (YFH1)

Saccharomyces cerevisiae cells lacking the yeast frataxin homologue (Δyfh1) accumulate iron in the mitochondria in the form of nanoparticles of ferric phosphate. The phosphate content of Δyfh1 mitochondria was higher than that of wild-type mitochondria, but the proportion of mitochondrial phosphate that was soluble was much lower in Δyfh1 cells. The rates of phosphate and iron uptake in vitro by isolated mitochondria were higher for Δyfh1 than wild-type mitochondria, and a significant proportion of the phosphate and iron rapidly became insoluble in the mitochondrial matrix, suggesting co-precipitation of these species after oxidation of iron by oxygen. Increasing the amount of phosphate in the medium decreased the amount of iron accumulated by Δyfh1 cells and improved their growth in an iron-dependent manner, and this effect was mostly transcriptional. Overexpressing the major mitochondrial phosphate carrier, MIR1, slightly increased the concentration of soluble mitochondrial phosphate and significantly improved various mitochondrial functions (cytochromes, [Fe-S] clusters, and respiration) in Δyfh1 cells. We conclude that in Δyfh1 cells, soluble phosphate is limiting, due to its co-precipitation with iron.

Protective effect of the dimer of 16,16-diMePGB1 against KCN-induced mitochondrial failure in hepatocytes.

The dimer and trimer of 16,16-dimethyl-15-dehydroprostaglandin B1 (16,16-diMePGB1) previously have been shown to have protective effects on mitochondrial function. To examine the potential mechanisms involved in protection against mitochondrial failure, we have studied the effects of the dimer of 16,16-diMe-PGB1 (dicalciphor) on mitochondrial function in hepatocytes exposed to KCN. Addition of micromolar concentrations of dicalciphor provided substantial protection against KCN-induced toxicity in a concentration- and time-dependent manner. Dicalciphor, however, had no effect on total or mitochondrial ATP losses in KCN-treated cells. The dimer prevented the marked loss of mitochondrial membrane potential (delta psi) and delta pH that occurs as a result of KCN treatment and prevented KCN-induced loading of phosphate in mitochondria. Furthermore, the dimer of 16,16-diMePGB1 also prevented KCN-induced mitochondrial and cellular swelling. These results demonstrate that dicalciphor protects against KCN-induced damage and that this protection is associated with regulation of specific mitochondrial ion transport functions.

Arginine-specific carbamoyl phosphate metabolism in mitochondria of Neurospora crassa. Channeling and control by arginine.

Citrulline is synthesized in mitochondria of Neurospora crassa from ornithine and carbamoyl phosphate. In mycelia grown in minimal medium, carbamoyl phosphate limits citrulline (and arginine) synthesis. Addition of arginine to such cultures reduces the availability of intramitochondrial ornithine, and ornithine then limits citrulline synthesis. We have found that for some time after addition of excess arginine, carbamoyl phosphate synthesis continued. Very little of this carbamoyl phosphate escaped the mitochondrion to be used in the pyrimidine pathway in the nucleus. Instead, mitochondrial carbamoyl phosphate accumulated over 40-fold and turned over rapidly. This was true in ornithine- or ornithine carbamoyltransferase-deficient mutants and in normal mycelia during feedback inhibition of ornithine synthesis. The data suggest that the rate of carbamoyl phosphate synthesis is dependent to a large extent upon the specific activity of the slowly and incompletely repressible synthetic enzyme, carbamoyl-phosphate synthetase A. In keeping with this conclusion, we found that when carbamoyl-phosphate synthetase A was repressed 2-10-fold by growth of mycelia in arginine, carbamoyl phosphate was still synthesized in excess of that used for residual citrulline synthesis. Again, only a small fraction of the excess carbamoyl phosphate could be accounted for by diversion to the pyrimidine pathway. The continued synthesis and turnover of carbamoyl phosphate in mitochondria of arginine-grown cells may allow rapid resumption of citrulline formation after external arginine disappears and no longer exerts negative control on ornithine biosynthesis.

Phosphocitrate inhibits mitochondrial and cytosolic accumulation of calcium in kidney cells in vivo.

Synthetic 3-phosphocitrate, an extremely potent inhibitor of calcium phosphate crystallization as determined in a nonbiological physical-chemical assay, has many similarities to a mitochondrial factor that inhibits crystallization of nondiffracting amorphous calcium phosphate. In order to determine whether phosphocitrate can prevent uptake and crystallization of calcium phosphate in mitochondria in vivo, it was administered intraperitoneally to animals given large daily doses of calcium gluconate or parathyroid hormone, a regimen that causes massive accumulation and crystallization of calcium phosphate in the mitochondria and cytosol of renal tubule cells in vivo. Administration of phosphocitrate greatly reduced the net uptake of Ca2+ by the kidneys and prevented the appearance of apatite-like crystalline structures within the mitochondrial matrix and cytosol of renal tubule cells. Phosphocitrate, which is a poor chelator of Ca2+, did not reduce the hypercalcemia induced by either agent. These in vivo observations therefore indicate that phosphocitrate acts primarily at the cellular level to prevent the extensive accumulation of calcium phosphate in kidney cells by inhibiting the mitochondrial accumulation or crystallization of calcium phosphate.

Ecdysone-inducible functions of larval fat bodies in Drosophila

Ecdysone-inducible functions of larval fat bodies in <i>Drosophila</i>

Incorporation of 32Pi into pyruvate dehydrogenase phosphate in mitochondria from control and insulin-treated adipose tissue

Incorporation of 32Pi into pyruvate dehydrogenase phosphate in mitochondria from control and insulin-treated adipose tissue

Phosphate transport in rat liver mitochondria. Membrane components labeled by N-ethylmaleimide during inhibition of transport.

N-ethylmaleimide (NEM) inhibits the transport of phosphate in mitochondria but is without effect on permeation of other metabolities. In spite of its specificity for inhibition of phosphate transport, NEM reacts in an unspecific manner with inner membrane proteins in general. Treatment of mitochondria with [3H]NEM just sufficient to produce inhibition of phosphate transport results in labeling of at least 10 polypeptide components of the inner membrane. A marked increase in the specificity of reaction of NEM for components of the phosphate transport system is attained by first protecting the transport system with p-mercuribenzoate (p-MB) and then by irreversibly blocking reactive sulfhydryl groups unassociated with transport by the addition of unlabeled NEM. Subsequent addition of dithiothreitol removes p-MB and restores 65 to 75 percent of the original phosphate transport activity. Reinhibition of transport with [3H]NEM results in both a 6-fold decrease in the amount of [3H]NEM bound by purified inner membrane vesicles and a substantial reduction in the number of labeled polypeptide components. Five distinct labeled species are detected by this method, one of which is a 32,000 molecular weight protein containing 40 percent of the bound radioactivity, or approximately 160 pmol/mg of inner membrane protein. Correlation of binding of [3H]NEM by inner membrane proteins with inhibition of phosphate transport suggests that the maximum concentration of the NEM-sensitive component of the phosphate transport system is 60 pmol/mg of mitochondrial protein. This value, when combined with V-max of NEM-sensitive transport of 205 nmol times min-1 times mg-1 at O degrees (Coty, W. A., and Pedersen, P. L. (1974) J. Biol. Chem. 249, 2593) yields an approximate minimum turnover for this process of 3500 min-1 at 0 degrees. This turnover number is at least 20-fold greater than similarly calculated values for adenine nucleotide transport and succinate oxidation in rat liver mitochondria at this temperature. Taken together these results suggest that the NEM-sensitive phosphate transport system in rat liver mitochondria has an unusually high catalytic activity compared to other mitochondrial processes, and that at least one of the five NEM-binding proteins is likely to be an essential component of this transport system.

New aspects of the mechanism of inorganic phosphate and dicarboxylate transport in mitochondria

1. Introduction It is generally accepted that the transport of inor- ganic phosphate in mitochondria is mediated by two distinct translocating systems localized in the inner membrane [l-7]. The first mediates the exchange- diffusion of Pi with OH-(phosphate carrier) and the second that of Pi with dicarboxylates (dicarboxyla- te carrier). The observation that Pi -Pi exchange is inhibited when both N-ethylmaleimide (NEM) and butylmalonate (BM) are present [4,7], together with the finding that dicarboxylate-dicarboxylate or Pi- dicarboxylate exchanges are not inhibited by NEM [8,9], are considered as main evidence that the two carriers are well distinct functional entities. We have however, found that butymalonate interfers with the mechanism of action of NEM since it removes or pre- vents in intact mitochondria the extrabinding of Pi in- duced by NEM [lo]. These findings have prompted us to reinvestigate the effect of NEM and BM on the transport of Pi and dicarboxylic acids in rat-liver mito- chondria. The results reported in this paper indicate that the mitochondrial membrane contains only one transporting system which, depending on the prevailing conditions, mediates either Pi-OH-or Pi-dicarboxy- late exchange. 2. Methods Rat-liver mitochondria were loaded with Pi, malate or succinate by preincubation for 10 min at 4” C in the presence of 250 mM sucrose, 5 I.cg/ml oligomycin, 0.34 m 200 mM sucrose; 20 mM Tris-HCI; oligomycin; rotenone and antimycin at the concentrations indicated above. The pH was ad- justed to 7.2. At the time specified in the legends nige- kin, butylmalonate, N-ethylmaleimide and the coun- ter-anions were added. Incubation was carried out at 4°C directly in small centrifuge tubes in a total vol of 1 ml and mitochondria separated from the suspen- ding medium by rapid centrifugation at 20 000 g. Before incubation Pi was determined chemically [ 1 l] and malate enzymatically [ 121. Their specific activity was calculated by measuring the corresponding total radioactivity of 12% HC104 extracts of loaded mito- chondria. Pi, malate and [14C] succinate content of mitochondrial pellet was corrected for that present in the sucrose space. 3. Results The sensitivity of the Pi carrier to NEM and BM was studied by following the efflux of P, from mito- chondria induced by the addition of nigericin [6]. Fig. 1 shows that NEM at 200 PM fully inhibited the Pi-OH-exchange whilst BM, even at a very high con- centration as 5 mM (not shown), had no effect at all. However when these two compounds were added to- gether different results, according to the sequence of additions and NEM concentration, were obtained. BM, added after NEM, increased the inhibition of Pi efflux

VOLUME CHANGES INDUCED BY INORGANIC PHOSPHATE IN LIVER MITOCHONDRIA.

VOLUME CHANGES INDUCED BY INORGANIC PHOSPHATE IN LIVER MITOCHONDRIA.