Mitochondrial Membrane Vesicles Research Articles

The mitochondrial ADP/ATP carrier exists and functions as a monomer.

For more than 40 years, the oligomeric state of members of the mitochondrial carrier family (SLC25) has been the subject of debate. Initially, the consensus was that they were dimeric, based on the application of a large number of different techniques. However, the structures of the mitochondrial ADP/ATP carrier, a member of the family, clearly demonstrated that its structural fold is monomeric, lacking a conserved dimerisation interface. A re-evaluation of previously published data, with the advantage of hindsight, concluded that technical errors were at the basis of the earlier dimer claims. Here, we revisit this topic, as new claims for the existence of dimers of the bovine ADP/ATP carrier have emerged using native mass spectrometry of mitochondrial membrane vesicles. However, the measured mass does not agree with previously published values, and a large number of post-translational modifications are proposed to account for the difference. Contrarily, these modifications are not observed in electron density maps of the bovine carrier. If they were present, they would interfere with the structure and function of the carrier, including inhibitor and substrate binding. Furthermore, the reported mass does not account for three tightly bound cardiolipin molecules, which are consistently observed in other studies and are important stabilising factors for the transport mechanism. The monomeric carrier has all of the required properties for a functional transporter and undergoes large conformational changes that are incompatible with a stable dimerisation interface. Thus, our view that the native mitochondrial ADP/ATP carrier exists and functions as a monomer remains unaltered.

The yeast mitochondrial carrier proteins Mrs3p/Mrs4p mediate iron transport across the inner mitochondrial membrane

The yeast proteins Mrs3p and Mrs4p are two closely related members of the mitochondrial carrier family (MCF), which had previously been implicated in mitochondrial Fe 2+ homeostasis. A vertebrate Mrs3/4 homologue named mitoferrin was shown to be essential for erythroid iron utilization and proposed to function as an essential mitochondrial iron importer. Indirect reporter assays in isolated yeast mitochondria indicated that the Mrs3/4 proteins are involved in mitochondrial Fe 2+ utilization or transport under iron-limiting conditions. To have a more direct test for Mrs3/4p mediated iron uptake into mitochondria we studied iron (II) transport across yeast inner mitochondrial membrane vesicles (SMPs) using the iron-sensitive fluorophore PhenGreen SK (PGSK). Wild-type SMPs showed rapid uptake of Fe 2+ which was driven by the external Fe 2+ concentration and stimulated by acidic pH. SMPs from the double deletion strain mrs3/4Δ failed to show this rapid Fe 2+ uptake, while SMPs from cells overproducing Mrs3/4p exhibited increased Fe 2+ uptake rates. Cu 2+ was transported at similar rates as Fe 2+, while other divalent cations, such as Zn 2+ and Cd 2+ apparently did not serve as substrates for the Mrs3/4p transporters. We conclude that the carrier proteins Mrs3p and Mrs4p transport Fe 2+ across the inner mitochondrial membrane. Their activity is dependent on the pH gradient and it is stimulated by iron shortage.

Identification of mitochondrial F1F0‐ATP synthase interacting with galectin‐3 in colon cancer cells

To evaluate the effect of galectin-3 in cell cycle regulation of colon cancer cells, we looked for binding molecules interacting with galectin-3 and examined the changes in cell cycle by suppressing galectin-3 and the binding molecule. To identify target molecules interacting with galectin-3, we analyzed immunoprecipitate of the anti-galectin-3 antibody obtained from human colon cancer cell line, using matrix-assisted laser desorption ionization-mass spectrometry. We validated subcellular localization of galectin-3 and ATP synthase identified, and ATP synthase activity was determined in the presence of galectin-3. Cell cycle regulation was monitored after galectin-3 siRNA transfection. ATP synthase b-subunit was identified in immunoprecipitate of the anti-galectin-3 antibody. Galectin-3 and ATP synthase were co-isolated in the inner membrane vesicles of mitochondria. Galectin-3 has an inhibitory activity against ATP synthase, and intracellular ATP content showed increasing tendency after galectin-3 suppression. Suppression of galectin-3 resulted in G0/G1 progression of human colon cancer cells arrested at S, S/G2 and G2/M phase in the presence of doxorubicin, and etoposide or nocodazole, respectively. Compared to cells in which ATP synthase d-subunit was suppressed alone, sub-G1 fraction caused by etoposide or nocodazole was decreased in cells with galectin-3 suppression alone. In conclusion, galectin-3 co-localized with ATP synthase in the inner membrane of mitochondria and has an inhibitory effect on ATP synthase in human colon cancer cells. In the presence of cell cycle synchronizing drugs, doxorubicin, etoposide, or nocodazole, suppression of galectin-3 induced cell cycle progression to G0/G1 phase.

Subunit 9 of the mitochondrial ATP synthase of Trypanosoma brucei is nuclearly encoded and developmentally regulated

We have previously shown that the mitochondrial ATP synthase is developmentally regulated through the life cycle of Trypanosoma brucei. The mechanism of this regulation is as yet unknown. We are currently examining regulation of expression of several key subunits of the ATP synthase to investigate this mechanism. In the work presented here, we have cloned, sequenced, and confirmed the identity of the ATPase subunit 9 homologue from T. brucei. The ATPase subunit 9 gene that we have identified from T. brucei has between 40 and 60% identity with subunit 9 from a variety of organisms. This gene possesses a putative mitochondrial import sequence at the N terminus of the encoded protein sequence. The protein expressed from this gene by in vitro transcription/translation comigrates with native protein isolated from inner mitochondrial membrane vesicles from T. brucei. We have shown that the cDNA identifies a copy of this gene in the nuclear genome, but does not identify a similar gene in kinetoplast DNA (kDNA) prepared from T. brucei. This gene does not show homology to any published sequence data from maxicircle DNA or edited maxicircle derived sequences. Steady state transcripts of a single size have been identified by Northern analysis and demonstrate significant developmental regulation through the T. brucei life cycle. Northern analysis and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) results show that the transcript is 10–14-fold higher in procyclic form than in early and late bloodstream forms.

FLX1 Codes for a Carrier Protein Involved in Maintaining a Proper Balance of Flavin Nucleotides in Yeast Mitochondria

Respiratory defective mutants of Saccharomyces cerevisiae previously assigned to complementation group G178 are characterized by an abnormally low ratio of FAD/FMN in mitochondria. A nuclear gene, designated FLX1, was selected from a yeast genomic library, based on its ability to confer wild-type growth properties to a representative G178 mutant. Genetic evidence has confirmed that the flavin nucleotide imbalance of G178 mutants is caused by mutations in FLX1. The sequence of FLX1 is identical to a reading frame recently reported to be present on yeast chromosome IX (GenBank Z47047). The sequence and tripartite repeat structure of the FLX1 product (Flx1p) indicate it is a member of a protein family consisting of mitochondrial substrate and nucleotide carriers. In yeast, FAD synthetase is present in the soluble cytoplasmic protein fraction but not in mitochondria. Riboflavin kinase, the preceding enzyme in flavin biosynthesis, is present in both subcellular fractions. The absence of FAD synthetase in mitochondria implies that FAD is imported from the cytoplasm. The lower concentration of mitochondrial FAD in flx1 mutants suggests that Flx1p is involved in flavin transport, a role that is also supported by biochemical evidence indicating more efficient flux of FAD across mitochondrial membrane vesicles prepared from wild-type strains than membrane vesicles from flx1 mutants.

Identification of a GTP-binding protein in the contact sites between inner and outer mitochondrial membranes

Whilst investigating whether GTP hydrolysis may be required for the import of preproteins into mitochondria we have found that a GTP-binding protein is located at the contact sites between mitochondrial inner and outer membranes. When mitochondrial outer membranes purified from rat liver were UV-irradiated in the presence of [alpha-32P]GTP, a 52 kDa protein was radiolabelled, whereas [alpha-32P]ATP did not label this protein. GTP-binding proteins were also labelled in the cytosolic and microsomal fractions, but the 52 kDa protein was concentrated in mitochondrial membranes and was the only protein specifically labelled by GTP in these membranes. Fractionation of mitochondrial membrane vesicles into outer membranes, inner membranes and contact sites between outer and inner membranes showed that the GTP-binding activity was highly enriched in contact sites, the location at which preprotein import is believed to occur. A protein of almost identical size was also found to be labelled in mitochondria from yeast.

Oxidative Phosphorylation Coupled to Respiration in Highly Purified Intact Euglena Mitochondria

Summary Improved methods for isolation of mitochondria from Euglena gracilis var. bacillaris are presented based on Yeda press breakage of protease-prepared sphaeroplasts of low vitamin B12 grown cells. Active preparations are obtained from dark-grown cells of wild-type or mutants W3BUL or W10BSmL but W1o is preferred since it lacks proplastids that can contaminate the mitochondrial preparations. Crude mitochondria obtained by differential centrifugation are further purified on Percoll gradients yielding five fractions (in order of decreasing density): A) enriched in clean contracted mitochondria; B) enriched in clean orthodox mitochondria; and C) D) and E) which contain broken mitochondria and mitochondrial membrane vesicles. All fractions show succinic dehydrogenase activity but only fractions A and B are intact based on retention of fumarase activity. Fraction A contains the bulk of the protein and consistently shows the highest respiratory activity, the activity of fraction B is somewhat lower and resembles the activity of crude mitochondria; fractions C, D and E are inactive. Oxygen uptake (unstimulated by ADP) by fraction A mitochondria is inhibited 100 % by 0.1 mM KCN, about 30 % by 6.6 μM antimycin A and about 50 % by 5 mM NaN3. True uncoupling is observed for the first time in Euglena mitochondria since dinitrophenol (DNP) (0.16 mM) and carbonylcyanide mtrichlorophenylhydrazone (m-CCCP) (0.2 μM) increase O2 uptake by about 250 %. P to O ratios of 2 - 3 are obtained. Electron microscopy and plating on rich media indicate that these preparations are free of significant contamination with other cell fractions or with microorganisms.

Isolation and reconstitution of an N-ethylmaleimide-sensitive phosphate transport protein from rat liver mitochondria

An N-ethylmaleimide-sensitive phosphate transport protein has been isolated from rat liver mitochondria, substantially purified, and reconstituted into phospholipid vesicles. Purified inner mitochondrial membrane vesicles depleted of F 1-ATPase by urea treatment proved to be the most satisfactory starting material. Treatment of these membrane vesicles with Triton X-100 resulted in solubilization of the phosphate transport protein. Further purification was achieved using hydroxylapatite powder. Polyacrylamide gel electrophoresis of the purified fraction in sodium dodecyl sulfate indicated the presence of two Coomassie blue-staining bands with apparent M r' s of 30,000 and 35,000. Labeling of the 35,000 M r band by the P i transport inhibitor diazobenzene sulfonate was reduced markedly by prior treatment of the mitochondria with the inhibitor N-ethylmaleimide. The purified fraction containing both proteins could be reconstituted into liposomes prepared from purified asolectin. Phosphate efflux from these vesicles was inhibited by N-ethylmaleimide, by the impermeant mercurial agent, p-chloromercuribenzoate, and by diazobenzene sulfonate. Treatment of the purified fraction with N-ethylmaleimide prior to incorporation into liposomes resulted in a reconstituted system incapable of catalyzing P i efflux. These studies summarize the first detailed attempt to purify the P i H + transport system from rat liver mitochondria and emphasize the need to commence the purification with purified inner membrane vesicles depleted of F 1-ATPase. In addition, these studies show that the final fraction contains a reconstitutively active transport system which when incorporated into phospholipid vesicles has its essential sulfhydryl groups oriented outward. Finally, it is shown that the purified fraction also contains a 30,000 M r component.

Substrate-selective activation of rat liver mitochondrial mono amine oxidase by oxygen

The activation by oxygen of the activity of monoamine oxidase (MAO) in preparations of rat liver mitochondrial membrane vesicles has been studied. The increase of activity with the substrates 5-hydroxytryptamine, tyramine, β-phenethylamine and benzylamine were in all cases uncompetitive. However, the degree of activation depended upon the particular substrate used. An attempt has been made to explain these results by use of an assumption that MAO activity can be divided into two forms unrelated to MAO-A and MAO-B. These two forms, tentatively called MAO-1 and MAO-2, differ in their Michaelis constants for oxygen.