Inactivation of mitochondrial complex I stimulates chloroplast ATPase in Physcomitrium patens

Light is the ultimate source of energy for photosynthetic organisms, but respiration is fundamental for supporting metabolism during the night or in heterotrophic tissues. In this work, we isolated Physcomitrella (Physcomitrium patens) plants with altered respiration by inactivating Complex I (CI) of the mitochondrial electron transport chain by independently targeting on two essential subunits. Inactivation of CI caused a strong growth impairment even in fully autotrophic conditions in tissues where all cells are photosynthetically active, demonstrating that respiration is essential for photosynthesis. CI mutants showed alterations in the stoichiometry of respiratory complexes while the composition of photosynthetic apparatus was substantially unaffected. CI mutants showed altered photosynthesis with high activity of both Photosystems I and II, likely the result of high chloroplast ATPase activity that led to smaller ΔpH formation across thylakoid membranes, decreasing photosynthetic control on cytochrome b6f in CI mutants. These results demonstrate that alteration of respiratory activity directly impacts photosynthesis in P. patens and that metabolic interaction between organelles is essential in their ability to use light energy for growth.

Mosses lacking mitochondrial Complex I show induced activation of chloroplast ATPase, demonstrating the impact of respiration on the metabolism of plant photosynthetic cells.     

A small ATPase protein of Arabidopsis, TGD3, involved in chloroplast lipid import

Polar lipid trafficking is essential in eukaryotic cells as membranes of lipid assembly are often distinct from final destination membranes. A striking example is the biogenesis of the photosynthetic membranes (thylakoids) in plastids of plants. Lipid biosynthetic enzymes at the endoplasmic reticulum and the inner and outer plastid envelope membranes are involved. This compartmentalization requires extensive lipid trafficking. Mutants of Arabidopsis are available that are disrupted in the incorporation of endoplasmic reticulum-derived lipid precursors into thylakoid lipids. Two proteins affected in two of these mutants, trigalactosyldiacylglycerol 1 (TGD1) and TGD2, encode the permease and substrate binding component, respectively, of a proposed lipid translocator at the inner chloroplast envelope membrane. Here we describe a third protein of Arabidopsis, TGD3, a small ATPase proposed to be part of this translocator. As in the tgd1 and tgd2 mutants, triacylglycerols and trigalactolipids accumulate in a tgd3 mutant carrying a T-DNA insertion just 5' of the TGD3 coding region. The TGD3 protein shows basal ATPase activity and is localized inside the chloroplast beyond the inner chloroplast envelope membrane. Proteins orthologous to TGD1, -2, and -3 are predicted to be present in Gram- bacteria, and the respective genes are organized in operons suggesting a common biochemical role for the gene products. Based on the current analysis, it is hypothesized that TGD3 is the missing ATPase component of a lipid transporter involving TGD1 and TGD2 required for the biosynthesis of ER-derived thylakoid lipids in Arabidopsis.     

Mitoskelin: a mitochondrial protein found in cytoskeletal preparations

A 70 kD protein, which we have named mitoskelin, is highly enriched in cytoskeletal preparations from bovine cardiac muscle. Mitoskelin has three main variants with isoelectric points between 5.6 and 5.8. Immunoblotting with polyclonal antibodies directed against mitoskelin shows that, like intermediate filament proteins, the majority of mitoskelin resists solubilization from a myocardial homogenate by a series of extraction solutions ranging from very low salt to 0.6 M KI buffers and by 0.1-1% Nonidet P-40 detergent. By double-label immunofluorescence on cells and tissues, mitoskelin is colocalized with the mitochondrial marker cytochrome c oxidase. Mitoskelin is associated with the inner membranes of mitochondria as shown by immunoelectron microscopy and immunoblotting. Immunological cross-reactivity and similarities of molecular weight, pI, distribution, and chromatographic properties indicate that mitoskelin is the 70 kD component of complex I (NADH: ubiquinone oxidoreductase), a portion of the mitochondrial oxidative phosphorylation system. No function or activity has yet been demonstrated for the 70 kD component of the 25-polypeptide complex I. Dialysis against physiological buffers allows purified, urea-solubilized mitoskelin to form 10 nm wide filamentous structures that do not closely resemble intermediate filaments. These results suggest the exciting possibility that mitochondria may contain a membrane-associated filamentous skeleton.     

Antihypertensive compounds with potent protein kinases inhibitory activity

A new indorocarbazole antibiotic SF2370 has been found in the culture broth of Actinomadura sp.; it has also been found to have the activities of antihypertension and diuresis. Derivatives of SF2370, NA0344, NA0345 and NA0346, when injected (0.1-1 mg/kg i.v.) significantly lowered the blood pressure of anesthetized normotensive rats and showed a long lasting antihypertensive action. In the case of p.o. administration to conscious restrained SHRs, measurement of blood pressure by the plethysmographic method indicated that 10 mg/kg of the compounds was enough to lower the blood pressure; the antihypertensive activities were found to remain more than 12 hrs after oral administration. By studying of the mode of antihypertensive action, these compounds dose dependently relaxed the isolated aortic preparation contracted by norepinephrine. In addition, it was found that protein kinase C activity of mice brain was inhibited by the compounds; the IC50 values were in the range of 0.062-0.20 microM. Moreover, a superprecipitation of actomyosin from smooth muscle of chicken gizzard was inhibited by the compounds, having an IC50 values of 0.31-0.72 microM.     

Effect of nucleotides and inhibitory anions on mitochondrial ATPase. Its pH dependence

The effect of pH on the sensitivity of F1-ATPase as well as mitochondrial ATPase activity to nucleoside diand triphosphates and to inhibitory anions such as cyanate and thiocyanate, has been studied. The results obtained show that nucleotides could act as activators or inhibitors of the ATPase hydrolytic activity depending on pH, substrate concentration, and binding of the enzyme to the membrane. The effect of those nucleotides which activate the hydrolysis of ATP-Mg2+ was more pronounced beyon the optimum pH corresponding to each of the three catalytic sites of the enzyme, whereas those which are inhibitors had a lower effect above this value. The sensitivity to the inhibitory anions decreased with increasing pH values; the decrease in the inhibitory effect was sharper when approaching the optimum pH value. These data are in agreement with the existence in mitochondrial ATPase of two different regulatory sites, one being specific for binding nucleotides, and another for anions. Both of them showed a different response upon changes of pH.     

A novel extranuclear mutant of Neurospora with a temperature-sensitive defect in mitochondrial protein synthesis and mitochondrial ATPase

Summary [C93] is a novel, extranuclear mutant of Neurospora crassa which has a normal mitochondrial phenotype when grown at 25°, but which is deficient in cytochromes b and aa ₃ when grown at 37° (Pittenger and West 1979). In the present work, the phenotype of [C93] was characterized in greater detail. When [C93] is grown at 37°, the rate of mitochondrial protein synthesis is decreased to approximately 25% that of wild type; the ratio of mitochondrial small to large ribosomal subunits is decreased to 1:4 and mitochondrial small subunits are deficient in the mitochondrially-synthesized protein, S-5. The mitochondrial ribosome assembly defects in 37°-grown [C93] resemble those in chloramphenicol-treated wild-type cells and could merely be a consequence of the decreased rates of mitochondrial protein synthesis. Analysis of mitochondrial translation products by SDS gel electrophoresis suggests that 37°-grown [C93] is grossly deficient in the 19,000 Mr subunit of the oligomycin-sensitive ATPase relative to other mitochondrially-synthesized proteins. The ATPase defect was not found in other extranuclear or nuclear mutants deficient in mitochondrial protein synthesis. These data and additional evidence suggest that the primary defect in [C93] may be in the assembly of the ATPase complex. The possible connection between the ATPase defect and the deficiency of mitochondrial protein synthesis is discussed.     

A temperature-dependent structural change of mitochondrial ATPase

The temperature dependence of the intrinsic tryptophan fluorescence in either bovine heart submitochondrial particles or oligomycin-sensitive ATPase isolated therefrom shows a discontinuity at near 25°C, which coincides with the temperature where a break in the Arrhenius plot of ATPase activity is found. Addition of n-butanol to submitochondrial particles induces a decrease of tryptophan fluorescence in the whole temperature range. The discontinuity is interpreted as a temperature-dependent structural change and related to a viscosity-induced phase separation of the intrinsic mitochondrial proteins.     

A family of repetitive palindromic sequences found in Neurospora mitochondrial DNA is also found in a mitochondrial plasmid DNA

Neurospora mtDNA contains a repetitive, 18 nucleotide palindromic sequence (5'-CCCTGCAGTACTGCAGGG-3') that contains two closely spaced PstI sites (CTGCAG) in the arms of the palindrome (Yin, S., Heckman, J., and RajBhandary, U. L. (1981) Cell 26, 325-332). In the present study, DNA sequence analysis was carried out to determine whether PstI palindromes are present in an apparently distinct genetic element, the 3.6-kilobase mitochondrial plasmid from Neurospora crassa strain Mauriceville-1c (FGSC 2225). The plasmid contains a cluster of closely spaced PstI sites extending over a 0.4-kilobase region (Collins, R. A., Stohl, L. L., Cole, M. D., and Lambowitz, A. M. (1981) Cell 24, 443-452). The DNA sequence shows that the cluster consists of eight PstI sites organized in five palindromic elements. Two of the elements are identical with the canonical sequence found in mtDNA, whereas the remaining three elements differ from the canonical sequence by a few nucleotides. The occurrence of the PstI palindromes in two otherwise unrelated DNA species is consistent with the hypothesis that they are related to mobile DNA sequences that either propagate or were once capable of propagating within mitochondria.     

The energy-linked conformational equilibrium in chloroplast ATPase

1. The capacity of chloroplast ATPase to drive energy-requiring processes increases with, but the maximal ΔpH maintained by ATPase is independent of the degree of modification by dithioerythritol.

2. After modification by dithioerythritol, the ATPase remains subject to an energy-linked equilibrium between active and inactive ATPase conformations. This results in an energetic threshold, the magnitude of which is independent of the degree of modification by dithioerythritol or the presence of P_i and/or ADP, but increases with medium pH.     

Mutation of a mitochondrial outer membrane protein affects chloroplast lipid biosynthesis

Lipid biosynthesis in plant cells is associated with various organelles, and maintenance of cell lipid homeostasis requires nimble regulation and coordination. In plants, environmental cues such as phosphate limitation require readjustment of the lipid biosynthetic machinery to substitute phospholipids by non-phosphorous glycolipids. Biosynthesis of the galactoglycerolipids predominant in plants proceeds by a constitutive and an alternative pathway that is known to be induced in response to phosphate deprivation. Plant lipid galactosyltransferases involved in both pathways are associated with the plastid envelope membranes and are encoded by nuclear genes. To identify mechanisms governing the activity of the alternative galactoglycerolipid pathway, a genetic suppressor screen was conducted in the background of the digalactolipid-deficient dgd1 mutant of Arabidopsis. A suppressor line that partially restored digalactoglycerolipid content in the dgd1 background carries a point mutation in a mitochondrial protein, which was tentatively designated DGD1 SUPPRESSOR 1 (DGS1). Presumed orthologs of this protein are present in plants, algae and fungi, but its molecular function is not yet known. In the dgd1 dgs1 double mutant, expression of nuclear genes encoding enzymes of the alternative galactoglycerolipid pathway is increased and hydrogen peroxide levels are elevated. This increase in hydrogen peroxide is proposed to be the reason for activation of the alternative pathway in the dgd1 dgs1 double mutant. Accordingly, hydrogen peroxide and treatments producing reactive oxygen also activate the alternative pathway in the wild-type. These results likely implicate the production of reactive oxygen in the regulation of the alternative galactoglycerolipid pathway in plants.     

Inhibition of yeast mitochondrial ATPase by concanavalin A

Concanavalin A binds to and inhibits enzyme activity of the energy transducing ATPase from yeast mitochondria. Activity loss is completely reversed by glucose or α-methyl-d-mannose. Concanavalin A reacts with the F₁ portion of the ATPase complex, suggesting that this enzyme unit may be a glycoprotein. A major concanavalin A binding site is associated with the largest subunit of the F₁ enzyme.     

A novel mitochondrial and chloroplast peptidasome, PreP

A novel mitochondrial and chloroplast peptidasome, the Presequence Protease (PreP) degrades organellar targeting peptides as well as other unstructured peptides up to 65 amino acid residues in length. PreP belongs to the pitrilysin oligopeptidase family (M16C) containing an inverted zinc-binding motif. The crystal structure of Arabidopsis thaliana PreP, AtPreP, refined at 2.1 ?, revealed a novel mechanism of proteolysis in which two halves of the enzyme connected by a hinge region enclose a large catalytic chamber opening and closing in response to peptide binding. Double knock-out mutant of AtPreP1 and AtPreP2 results in a severe phenotype, including decreased size and growth rate, chlorosis and organellar abnormalities, such as altered chloroplast starch content, partial loss of the integrity of the inner mitochondrial membrane and reduced mitochondrial respiration. PreP homologues are also present in yeast and humans. Interestingly, human PreP has been associated with Alzheimer's disease as it is responsible for degradation of amyloid-β peptide in brain mitochondria.     

A plant outer mitochondrial membrane protein with high amino acid sequence identity to a chloroplast protein import receptor

The conductivity of two photosynthetic protein–pigment complexes, a light harvesting 2 complex and a reaction center, was measured with an atomic force microscope capable of performing electrical measurements. Current–voltage measurements were performed on complexes embedded in their natural environment. Embedding the complexes in lipid bilayers made it possible to discuss the different conduction behaviors of the two complexes in light of their atomic structure.