The transport of metabolites, coenzymes, and ions across the mitochondrial inner membrane is still poorly understood. In most cases, membrane transport is facilitated by the so-called mitochondrial carrier proteins. The yeast Saccharomyces cerevisiae contains 35 members of the carrier family, but a function has been identified for only 13 proteins. Here, we investigated the yeast carrier Leu5p (encoded by the gene YHR002w) and its close human homologue Graves' disease protein. Leu5p is inserted into the mitochondrial inner membrane along the specialized import pathway used by carrier proteins. Deletion of LEU5 (strain ⌬leu5) was accompanied by a 15-fold reduction of mitochondrial coenzyme A (CoA) levels but did not affect the cytosolic CoA content. As a consequence, the activities of several mitochondrial CoA-dependent enzymes were strongly decreased in ⌬leu5 cells. Our in vitro and in vivo analyses assign a function to Leu5p in the accumulation of CoA in mitochondria, presumably by serving as a transporter of CoA or a precursor thereof. Expression of the Graves' disease protein in ⌬leu5 cells can replace the function of Leu5p, demonstrating that the human protein represents the orthologue of yeast Leu5p. The function of the human protein might not be directly linked to the disease, as antisera derived from patients with active Graves' disease do not recognize the protein after expression in yeast, suggesting that it does not represent a major autoantigen. The two carrier proteins characterized herein are the first components for which a role in the subcellular distribution of CoA has been identified.Mitochondria perform a variety of processes, such as oxidative phosphorylation, the citric acid cycle, the -oxidation of fatty acids, parts of the urea cycle, and the biosynthesis of heme and certain amino acids (13,14,61). The metabolic activity of mitochondria requires the rapid and highly specific exchange of molecules between the cytosol and the mitochondrial matrix space. To a large extent, this is facilitated by a family of transport proteins of the inner membrane, the so-called mitochondrial carrier proteins (for reviews, see references 20, 22, 31, 44, 45, and 67). Members of this family include proteins responsible for the exchange of ADP and ATP (termed AAC or ANT) and for the transport of, e.g., phosphate, citrate, carnitine, dicarboxylates, amino acids, flavin adenine dinucleotide (FAD), or protons. The biogenesis of carriers differs in various aspects from that of most other mitochondrial proteins (reviewed in references 2, 32, and 60). They lack an N-terminal targeting sequence (presequence) and they follow a unique import pathway involving the interaction with specialized import components in the outer membrane (Tom70), the intermembrane space (Tim8, Tim9, Tim10, Tim12, and Tim13), and the inner membrane (Tim18, Tim22, and Tim54).In their transport-competent form, carrier proteins are dimeric (50). Each monomer is comprised of three homologous modules containing two transmembrane segments each. Both ...
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A c c e p t e d M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Garcia-Martinez EM et al., 2 AbstractMinocycline, an antibiotic of the tetracycline family, has attracted considerable interest for its theoretical therapeutic applications in neurodegenerative diseases. However, the mechanism of action underlying its effect remains elusive. Here we have studied the effect of minocycline under excitotoxic conditions. Fluorescence and bioluminescence imaging studies in rat cerebellar granular neuron cultures using fura-2/AM and mitochondria-targeted aequorin revealed that minocycline, at concentrations higher than those shown to block inflammation and inflammation-induced neuronal death, inhibited NMDA-induced cytosolic and mitochondrial rises in Ca 2+ concentrations in a reversible manner. Moreover, minocycline added in the course of NMDA stimulation decreased Ca 2+ intracellular levels, but not when induced by depolarization with a high K + medium. We also found that minocycline, at the same concentrations, partially depolarized mitochondria by about 5-30 mV, prevented mitochondrial Ca 2+ uptake under conditions of environmental stress, and abrogated NMDAinduced reactive oxygen species (ROS) formation. Consistently, minocycline also abrogates the rise in ROS induced by 75 M Ca 2+ in isolated brain mitochondria. In search for the mechanism of mitochondrial depolarization, we found that minocycline markedly inhibited state 3 respiration of rat brain mitochondria, although distinctly increased oxygen uptake in state 4. Minocycline inhibited NADH-cytochrome c reductase and cytochrome c oxidase activities, whereas the activity of succinate-cytochrome c reductase was not modified, suggesting selective inhibition of complex I and IV. Finally, minocycline affected activity of voltage-dependent anion channel (VDAC) as determined in the reconstituted system. Taken together, our results indicate that mitochondria are a critical factor in minocycline-mediated neuroprotection.
Methadone is a widely used therapeutic opioid in narcotic addiction and neuropathic pain syndromes. Oncologists regularly use methadone as a long-lasting analgesic. Recently it has also been proposed as a promising agent in leukemia therapy, especially when conventional therapies are not effective. Nevertheless, numerous reports indicate a negative impact on human cognition with chronic exposure to opiates. Thus, clarification of methadone toxicity is required. In SH-SY5Y cells we found that high concentrations of methadone were required to induce cell death. Methadone-induced cell death seems to be related to necrotic processes rather than typical apoptosis. Cell cultures challenged with methadone presented alterations in mitochondrial outer membrane permeability. A mechanism that involves Bax translocation to the mitochondria was observed, accompanied with cytochrome c release. Furthermore, no participation of known protein regulators of apoptosis such as Bcl-X(L) and p53 was observed. Interestingly, methadone-induced cell death took place by a caspases-independent pathway; perhaps due to its ability to induce a drastic depletion in cellular ATP levels. Therefore, we studied the effect of methadone on isolated rat liver mitochondria. We observed that methadone caused mitochondrial uncoupling, coinciding with the ionophoric properties of methadone, but did not cause swelling of the organelles. Overall, the effects observed for cells in the presence of supratherapeutic doses of methadone may result from a "bioenergetic crisis." A decreased level of cellular energy may predispose cells to necrotic-like cell death.
Although gradual deterioration of life functions with age is not a fundamental rule, it is pervasive among living organisms, regardless of their mode of reproduction and the number of constituent cells. However, deterioration can be temporarily arrested or slowed down due to the process of anhydrobiosis. Two modes of anhydrobiosis can be distinguished for the developmental and adult stages of animals. Developmental resting stages are reported for different animals, including sponges (Porifera), stingers (Cnidaria), flatworms (Platyhelminthes), insects (Insecta), copepods (Copepoda) and branchiopods (Branchiopoda). However, anhydrobiosis occurring at any stage of animal life, including adults, is found only in a few invertebrate phyla, namely roundworms (Nematoda), wheel animals (Rotifera) and water bears (Tardigrada). Notably, in the second group anhydrobiosis has been proposed to eliminate or slow‐down aging symptoms. This, in turn, may correlate with higher fitness and fecundity, and increased offspring longevity. We present available data concerning anhydrobiosis of tardigrades, bdelloid rotifers and nematodes, the only animals known to be capable of anhydrobiosis as adult individuals. The impact of anhydrobiosis on animal aging is illustrated by two models based on experimental data, namely the “Sleeping Beauty” and “Picture of Dorian Grey” models. According to the “Sleeping Beauty” model, anhydrobiotic organisms do not age during anhydrobiosis, whereas the “Picture of Dorian Grey” model predicts that the anhydrobiotic organism ages, at least during the initial stage of anhydrobiosis. Finally, we discuss possible implications of these models for individual longevity and survival as well as phenotypic diversity of taxa and their evolution. A better understanding of life strategies of anhydrobiotic animals both at the ontogenetic and phylogenetic levels can provide answers to many fundamental questions and useful practical outputs in branches of applied sciences.
The yeast Saccharomyces cerevisiae genome is endowed with two distinct isoforms of Voltage-Dependent Anion Channel (VDAC). The isoform yVDAC2 is currently understudied with respect to the best known yVDAC1. Yet, since the discovery, the function of yVDAC2 was unclear, leading to the hypothesis that it might be devoid of a channel function. In this work we have elucidated, by bioinformatics modeling and electrophysiological analysis, the functional activity of yVDAC2. The conformation of yVDAC2 and, for comparison, of yVDAC1 were modeled using a multiple template approach involving mouse, human and zebrafish structures and both showed to arrange the sequences as the typical 19-stranded VDAC β-barrel. Molecular dynamics simulations showed that yVDAC2, in comparison with yVDAC1, has a different number of permeation paths of potassium and chloride ions. yVDAC2 protein was over-expressed in the S. cerevisiae cells depleted of functional yVDAC1 (Δpor1 mutant) and, after purification, it was reconstituted in artificial membranes (planar lipid bilayer (PLB) system). The protein displayed channel-forming activity and the calculated conductance, voltage-dependence and ion selectivity values were similar to those of yVDAC1 and other members of VDAC family. This is the first time that yVDAC2 channel features are detected and characterized.
The protein(s) responsible for metabolite transport through the outer membrane of the yeast Saccharomyces cerevisiae mitochondria depleted of mitochondrial porin (also known as voltage-dependent anion selective channel), termed here porin1, is (are) still unidentified. It is postulated that the transport may be supported by the protein import machinery of the outer membrane, the TOM complex (translocase of the outer membrane). We demonstrate here that in the absence of functional porin1, the blockage of the TOM complex by the fusion protein termed pb(2)-DHFR (consisting of the first 167 amino acids of yeast cytochrome b(2) preprotein connected to mouse dihydrofolate reductase) limits the access of external NADH to mitochondria. It was measured by the ability of the blockage to inhibit external NADH oxidation by the proper dehydrogenase located at the outer surface of the inner membrane. The inhibition depends on external NADH concentration and increases with decreasing amounts of the substrate. In the presence of 1 microg of pb(2)-DHFR per 50 microg of mitochondrial protein almost quantitative inhibition was observed when external NADH was applied at the concentration of 70 nmol per mg of mitochondrial protein. On the other hand, external NADH decreases the levels of pb(2)-DHFR binding at the trans site of the TOM complex in porin1-depleted mitochondria in a concentration-dependent fashion. Our data define an important role of the TOM complex in the transport of external NADH across the outer membrane of porin1-depleted mitochondria.
Metal hyperaccumulating plants should have extremely efficient defense mechanisms, enabling growth and development in a polluted environment. Brassica species are known to display hyperaccumulation capability. Brassica juncea (Indiana mustard) v. Malopolska plants were exposed to trace elements, i.e., cadmium (Cd), copper (Cu), lead (Pb), and zinc (Zn), at a concentration of 50 μM and were then harvested after 96 h for analysis. We observed a high index of tolerance (IT), higher than 90%, for all B. juncea plants treated with the four metals, and we showed that Cd, Cu, Pb, and Zn accumulation was higher in the above-ground parts than in the roots. We estimated the metal effects on the generation of reactive oxygen species (ROS) and the levels of protein oxidation, as well as on the activity and gene expression of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX). The obtained results indicate that organo-specific ROS generation was higher in plants exposed to essential metal elements (i.e., Cu and Zn), compared with non-essential ones (i.e., Cd and Pb), in conjunction with SOD, CAT, and APX activity and expression at the level of encoding mRNAs and existing proteins. In addition to the potential usefulness of B. juncea in the phytoremediation process, the data provide important information concerning plant response to the presence of trace metals.
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