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Frontiers in Plant Science 2019Mitochondria play a central role in the energy metabolism of plants. At the same time, they provide energy for plant stress responses. We here report a first view on the...
Mitochondria play a central role in the energy metabolism of plants. At the same time, they provide energy for plant stress responses. We here report a first view on the mitochondrial Oxidative Phosphorylation (OXPHOS) system of the halophile (salt tolerant) plant . Mitochondria were purified from suspension cultures of and for comparison of , a closely related glycophyte (salt sensitive) plant. Mitochondria were treated with digitonin and solubilized protein complexes were analyzed by 2D Blue native/SDS polyacrylamide gel electrophoresis. The OXPHOS systems of the two compared plants exhibit some distinct differences. mitochondria include a very abundant respiratory supercomplex composed of monomeric complex I and dimeric complex III. At the same time the complexes II and IV are of reduced abundance. The stability of the OXPHOS complexes was investigated by combined salt and temperature treatments of isolated mitochondria. ATP synthase (complex V) is of increased stability in . Also, the I + III supercomplex is present in high abundance during stress treatments. These results give insights into the mitochondrial contribution to the plant salt stress response.
PubMed: 31456813
DOI: 10.3389/fpls.2019.01010 -
MethodsX 2019Key mitochondrial processes are known to be widely conserved throughout the eukaryotic domain. However, the scarce availability of working materials may restrict the...
Key mitochondrial processes are known to be widely conserved throughout the eukaryotic domain. However, the scarce availability of working materials may restrict the assessment of such mitochondrial activities in several working models. Pollen tube mitochondrial studies represent one example of this, where tests have been often restricted due the physical impossibility of performing experiments with isolated mitochondria in enough quantities. Here we detail a method to measure mitochondrial respiratory chain activity and calcium transport in tobacco pollen tubes. ••
PubMed: 31406686
DOI: 10.1016/j.mex.2019.07.023 -
Frontiers in Microbiology 2019The twin-arginine translocation (Tat) system transports folded proteins across membranes of prokaryotes, plant plastids, and some mitochondria. According to blue-native...
The twin-arginine translocation (Tat) system transports folded proteins across membranes of prokaryotes, plant plastids, and some mitochondria. According to blue-native polyacrylamide gel electrophoresis after solubilization with digitonin, distinct interactions between the components TatA, TatB, and TatC result in two major TatBC-containing complexes in that can bind protein substrates. We now report the first detection of a TatABC complex that likely represents the state at which transport occurs. This complex was initially found when the photo cross-linking amino acid -benzoyl-l-phenylalanine (Bpa) was introduced at position I50 on the periplasmic side of the first trans-membrane domain of TatC. Cross-linking of TatC resulted in TatC-TatC-cross-links, indicating a close proximity to neighboring TatC in the complex. However, the new complex was not caused by cross-links but rather by non-covalent side chain interactions, as it was also detectable without UV-cross-linking or with an I50Y exchange. The new complex did not contain any detectable substrate. It was slightly upshifted relative to previously reported substrate-containing TatABC complexes. In the absence of TatA, an inactive TatBC complex was formed of the size of wild-type substrate-containing TatABC complexes, suggesting that TatB occupies TatA-binding sites at TatC. When substrate binding was abolished by point mutations, this TatBC complex shifted analogously to active TatABC complexes, indicating that a defect substrate-binding site further enhances TatB association to TatA-binding sites. Only TatA could shift the complex with an intact substrate-binding site, which explains the TatA requirement for substrate transport by TatABC systems.
PubMed: 31354642
DOI: 10.3389/fmicb.2019.01482 -
Biochimica Et Biophysica Acta.... Aug 2019Light drives photosynthesis. In plants it is absorbed by light-harvesting antenna complexes associated with Photosystem I (PSI) and photosystem II (PSII). As PSI and...
Light drives photosynthesis. In plants it is absorbed by light-harvesting antenna complexes associated with Photosystem I (PSI) and photosystem II (PSII). As PSI and PSII work in series, it is important that the excitation pressure on the two photosystems is balanced. When plants are exposed to illumination that overexcites PSII, a special pool of the major light-harvesting complex LHCII is phosphorylated and moves from PSII to PSI (state 2). If instead PSI is over-excited the LHCII complex is dephosphorylated and moves back to PSII (state 1). Recent findings have suggested that LHCII might also transfer energy to PSI in state 1. In this work we used a combination of biochemistry and (time-resolved) fluorescence spectroscopy to investigate the PSI antenna size in state 1 and state 2 for Arabidopsis thaliana. Our data shows that 0.7 ± 0.1 unphosphorylated LHCII trimers per PSI are present in the stroma lamellae of state-1 plants. Upon transition to state 2 the antenna size of PSI in the stroma membrane increases with phosphorylated LHCIIs to a total of 1.2 ± 0.1 LHCII trimers per PSI. Both phosphorylated and unphosphorylated LHCII function as highly efficient PSI antenna.
Topics: Arabidopsis; Digitonin; Energy Transfer; Light; Light-Harvesting Protein Complexes; Phosphorylation; Photosystem I Protein Complex; Photosystem II Protein Complex; Spectrometry, Fluorescence
PubMed: 31299182
DOI: 10.1016/j.bbabio.2019.07.001 -
The Journal of Biological Chemistry Jul 2019Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes. Although it has long been known that CL plays an important role in mitochondrial...
Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes. Although it has long been known that CL plays an important role in mitochondrial bioenergetics, recent evidence in the yeast model indicates that CL is also essential for intermediary metabolism. To gain insight into the function of CL in energy metabolism in mammalian cells, here we analyzed the metabolic flux of [U-C]glucose in a mouse C2C12 myoblast cell line, TAZ-KO, which is CL-deficient because of CRISPR/Cas9-mediated knockout of the CL-remodeling enzyme tafazzin (TAZ). TAZ-KO cells exhibited decreased flux of [U-C]glucose to [C]acetyl-CoA and M2 and M4 isotopomers of tricarboxylic acid (TCA) cycle intermediates. The activity of pyruvate carboxylase, the predominant enzyme for anaplerotic replenishing of the TCA cycle, was elevated in TAZ-KO cells, which also exhibited increased sensitivity to the pyruvate carboxylase inhibitor phenylacetate. We attributed a decreased carbon flux from glucose to acetyl-CoA in the TAZ-KO cells to a ∼50% decrease in pyruvate dehydrogenase (PDH) activity, which was observed in both TAZ-KO cells and cardiac tissue from TAZ-KO mice. Protein-lipid overlay experiments revealed that PDH binds to CL, and supplementing digitonin-solubilized TAZ-KO mitochondria with CL restored PDH activity to WT levels. Mitochondria from TAZ-KO cells exhibited an increase in phosphorylated PDH, levels of which were reduced in the presence of supplemented CL. These findings indicate that CL is required for optimal PDH activation, generation of acetyl-CoA, and TCA cycle function, findings that link the key mitochondrial lipid CL to TCA cycle function and energy metabolism.
Topics: Acetyl Coenzyme A; Acyltransferases; Animals; Carbon; Cardiolipins; Cell Line; Citric Acid Cycle; Energy Metabolism; Enzyme Activation; Lipids; Mice; Mice, Knockout; Mitochondria; Pyruvate Carboxylase; Pyruvate Dehydrogenase Complex; Transcription Factors
PubMed: 31186346
DOI: 10.1074/jbc.RA119.009037 -
The Journal of Biological Chemistry Jul 2019The mitochondrial F-ATP synthase is a complex molecular motor arranged in V-shaped dimers that is responsible for most cellular ATP synthesis in aerobic conditions. In...
The mitochondrial F-ATP synthase is a complex molecular motor arranged in V-shaped dimers that is responsible for most cellular ATP synthesis in aerobic conditions. In the yeast F-ATP synthase, subunits e and g of the F sector constitute a lateral domain, which is required for dimer stability and cristae formation. Here, by using site-directed mutagenesis, we identified Arg-8 of subunit e as a critical residue in mediating interactions between subunits e and g, most likely through an interaction with Glu-83 of subunit g. Consistent with this hypothesis, (i) the substitution of Arg-8 in subunit e (eArg-8) with Ala or Glu or of Glu-83 in subunit g (gGlu-83) with Ala or Lys destabilized the digitonin-extracted F-ATP synthase, resulting in decreased dimer formation as revealed by blue-native electrophoresis; and (ii) simultaneous substitution of eArg-8 with Glu and of gGlu-83 with Lys rescued digitonin-stable F-ATP synthase dimers. When tested in lipid bilayers for generation of Ca-dependent channels, WT dimers displayed the high-conductance channel activity expected for the mitochondrial megachannel/permeability transition pore, whereas dimers obtained at low digitonin concentrations from the Arg-8 variants displayed currents of strikingly small conductance. Remarkably, double replacement of eArg-8 with Glu and of gGlu-83 with Lys restored high-conductance channels indistinguishable from those seen in WT enzymes. These findings suggest that the interaction of subunit e with subunit g is important for generation of the full-conductance megachannel from F-ATP synthase.
Topics: Dimerization; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Mitochondrial Proton-Translocating ATPases; Mutagenesis, Site-Directed; Protein Stability; Protein Subunits; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 31160339
DOI: 10.1074/jbc.RA119.008775 -
Scientific Reports May 2019The membrane activity of some saponins, such as digitonin or alpha-hederin, is usually attributed to their interaction with membrane cholesterol (Chol). This contrasts...
The membrane activity of some saponins, such as digitonin or alpha-hederin, is usually attributed to their interaction with membrane cholesterol (Chol). This contrasts with our recent publication showing that Chol, contrary to sphingomyelin (SM), can delay the cytotoxicity of the saponin ginsenoside Rh2, challenging the usual view that most saponins mediate their membrane effects through interaction with Chol. The aim of the present study was to elucidate the respective importance of Chol and SM as compared to phosphatidylcholine (PC) species in the membrane-related effects of Rh2. On simple lipid monolayers, Rh2 interacted more favorably with eggSM and DOPC than with Chol and eggPC. Using Large Unilamellar Vesicles (LUVs) of binary or ternary lipid compositions, we showed that Rh2 increased vesicle size, decreased membrane fluidity and induced membrane permeability with the following preference: eggSM:eggPC > eggSM:eggPC:Chol > eggPC:Chol. On Giant Unilamellar Vesicles (GUVs), we evidenced that Rh2 generated positive curvatures in eggSM-containing GUVs and small buds followed by intra-luminal vesicles in eggSM-free GUVs. Altogether, our data indicate that eggSM promotes and accelerates membrane-related effects induced by Rh2 whereas Chol slows down and depresses these effects. This study reconsiders the theory that Chol is the only responsible for the activity of saponins.
Topics: Animals; Cell Membrane Permeability; Chickens; Cholesterol; Egg Proteins; Ginsenosides; Membrane Fluidity; Panax; Phosphatidylcholines; Sphingomyelins; Unilamellar Liposomes
PubMed: 31086211
DOI: 10.1038/s41598-019-43674-w -
Scientific Reports Apr 2019The plasma membrane of eukaryotic cells forms the essential barrier to the extracellular environment, and thus plasma membrane disruptions pose a fatal threat to cells....
The plasma membrane of eukaryotic cells forms the essential barrier to the extracellular environment, and thus plasma membrane disruptions pose a fatal threat to cells. Here, using invasive breast cancer cells we show that the Ca - and phospholipid-binding protein annexin A7 is part of the plasma membrane repair response by enabling assembly of the endosomal sorting complex required for transport (ESCRT) III. Following injury to the plasma membrane and Ca flux into the cytoplasm, annexin A7 forms a complex with apoptosis linked gene-2 (ALG-2) to facilitate proper recruitment and binding of ALG-2 and ALG-2-interacting protein X (ALIX) to the damaged membrane. ALG-2 and ALIX assemble the ESCRT III complex, which helps excise and shed the damaged portion of the plasma membrane during wound healing. Our results reveal a novel function of annexin A7 - enabling plasma membrane repair by regulating ESCRT III-mediated shedding of injured plasma membrane.
Topics: Annexin A7; Apoptosis Regulatory Proteins; Calcium-Binding Proteins; Cell Cycle Proteins; Cell Membrane; Digitonin; Endosomal Sorting Complexes Required for Transport; Female; HeLa Cells; Humans; MCF-7 Cells
PubMed: 31040365
DOI: 10.1038/s41598-019-43143-4 -
Plant Physiology Jul 2019The Antarctic psychrophile sp. UWO241 evolved in a permanently ice-covered lake whose aquatic environment is characterized not only by constant low temperature and high...
The Antarctic psychrophile sp. UWO241 evolved in a permanently ice-covered lake whose aquatic environment is characterized not only by constant low temperature and high salt but also by low light during the austral summer coupled with 6 months of complete darkness during the austral winter. Since the UWO241 genome indicated the presence of and protein kinases, we examined protein phosphorylation and the state transition phenomenon in this psychrophile. Light-dependent [γ-P]ATP labeling of thylakoid membranes from sp. UWO241 exhibited a distinct low temperature-dependent phosphorylation pattern compared to despite comparable levels of the Stt7 protein kinase. The sequence and structure of the UWO241 Stt7 kinase domain exhibits substantial alterations, which we suggest predisposes it to be more active at low temperature. Comparative purification of PSII and PSI combined with digitonin fractionation of thylakoid membranes indicated that UWO241 altered its thylakoid membrane architecture and reorganized the distribution of PSI and PSII units between granal and stromal lamellae. Although UWO241 grown at low salt and low temperature exhibited comparable thylakoid membrane appression to that of at its optimal growth condition, UWO241 grown under its natural condition of high salt resulted in swelling of the thylakoid lumen. This was associated with an upregulation of PSI cyclic electron flow by 50% compared to growth at low salt. Due to the unique 77K fluorescence emission spectra of intact UWO241 cells, deconvolution was necessary to detect enhancement in energy distribution between PSII and PSI, which was sensitive to the redox state of the plastoquinone pool and to the NaCl concentrations of the growth medium. We conclude that a reorganization of PSII and PSI in UWO241 results in a unique state transition phenomenon that is associated with altered protein phosphorylation and enhanced PSI cyclic electron flow. These data are discussed with respect to a possible PSII-PSI energy spillover mechanism that regulates photosystem energy partitioning and quenching.
Topics: Algal Proteins; Amino Acid Sequence; Antarctic Regions; Chlamydomonas; Chlamydomonas reinhardtii; Chlorophyll; Cold Temperature; Light; Microscopy, Electron, Transmission; Photosynthesis; Photosynthetic Reaction Center Complex Proteins; Protein Domains; Protein Kinases; Sequence Homology, Amino Acid; Species Specificity; Spectrometry, Fluorescence; Thylakoids
PubMed: 31019005
DOI: 10.1104/pp.19.00411 -
Scientific Reports Feb 2019Reprogramming of cultured cells using Xenopus egg extract involves controlling four major steps: plasma membrane permeabilization, egg factors import into the nucleus,...
Reprogramming of cultured cells using Xenopus egg extract involves controlling four major steps: plasma membrane permeabilization, egg factors import into the nucleus, membrane resealing, and cell proliferation. Using propidium iodide to assess plasma membrane permeability, we established that 90% of the cultured fin cells were permeabilized by digitonin without any cell losses. We showed that egg extract at metaphase II stage was essential to maintain nuclear import function in the permeabilized cells, as assessed with a fusion GFP protein carrying the nuclear import signal NLS. Moreover, the Xenopus-egg-specific Lamin B3 was detected in 87% of the cell nuclei, suggesting that other egg extract reprogramming factors of similar size could successfully enter the nucleus. Lamin B3 labelling was maintained in most cells recovered 24 h after membrane resealing with calcium, and cells successfully resumed cell cycle in culture. In contrast, permeabilized cells that were not treated with egg extract failed to proliferate in culture and died, implying that egg extract provided factor essential to the survival of those cells. To conclude, fish fin cells were successfully primed for treatment with reprogramming factors, and egg extract was shown to play a major role in their survival and recovery after permeabilization.
Topics: Animals; Cell Culture Techniques; Cells, Cultured; Cellular Reprogramming; Complex Mixtures; Goldfish; Ovum; Xenopus laevis
PubMed: 30814557
DOI: 10.1038/s41598-019-39500-y