[Show abstract][Hide abstract] ABSTRACT: Available data suggest that voltage-dependent anion selective channel (VDAC) constitutes an important component of a cellular regulatory mechanism based on the intracellular reduction/oxidation (redox) state. Here, using quantitative RT-PCR, we demonstrated that depletion of VDAC1 (termed here VDAC) in Saccharomyces cerevisiae cells distinctly affected levels of mRNAs encoding nuclear proteins sensitive to changes of the intracellular redox state including the nuclear transcription factors important for adaptation to the redox state and proteins involved in communication between mitochondria and the nucleus. We also revealed that the changes of the studied protein transcript levels generally correlated with changes of the intracellular redox state although VDAC appears also to affect mRNA levels by a mechanism not based on changes of the intracellular redox states. Thus, VDAC seems to be an important element of the intracellular signaling network.
Journal of Bioenergetics 11/2010; 42(6):483-9. · 1.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Proteins involved in apoptosis are still a matter of debate. Therefore, we decided to check the effect of the presence of VDAC (voltage dependent anion selective channel) on viability of Saccharomyces cerevisiae cells following their exposure to H(2)O(2) that is known to induce apoptosis both in S. cerevisiae and in mammalian cells. Mitochondria of S. cerevisiae contain only one channel-forming VDAC isoform (VDAC1), which simplifies studies on the channel. Using S. cerevisiae mutant depleted of VDAC1 (termed here VDAC) and the isogenic wild type, we have shown that VDAC is important for protection of S. cerevisiae cells against H(2)O(2) treatment, particularly in exponential growth phase that is known to be more affected by H(2)O(2). The increased viability of H(2)O(2) pretreated exponentially growing cells containing VDAC was accompanied by clear changes of the cytosol redox state and was potentiated by minocycline, an antibiotic of the tetracycline family that displays cytoprotective potency. The protective effect of minocycline also coincided with distinct changes of cytosol redox state. Thus, we conclude that the ability to change the cytosol redox state following exposure to H(2)O(2) or/and minocycline appears to be an intrinsic feature of exponentially growing cells (young cells) containing VDAC. Moreover, the ability seems to be crucial for both cell viability and protective effect of minocycline.
European journal of pharmacology 09/2010; 643(1):42-7. · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Available data indicate that superoxide anion (O(2)(*-) ) is released from mitochondria, but apart from VDAC (voltage dependent anion channel), the proteins involved in its transport across the mitochondrial outer membrane still remain elusive. Using mitochondria of the yeast Saccharomyces cerevisiae mutant depleted of VDAC (Deltapor1 mutant) and the isogenic wild type, we studied the role of the TOM complex (translocase of the outer membrane) in the efflux of O(2)(*-) from the mitochondria. We found that blocking the TOM complex with the fusion protein pb(2)-DHFR decreased O(2)(*-) release, particularly in the case of Deltapor1 mitochondria. We also observed that the effect of the TOM complex blockage on O(2)(*-) release from mitochondria coincided with the levels of O(2)(*-) release as well as with levels of Tom40 expression in the mitochondria. Thus, we conclude that the TOM complex participates in O(2)(*-) release from mitochondria.
Journal of Bioenergetics 09/2009; 41(4):361-7. · 1.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It is suggested that in the course of the TOM complex evolution at least two lineages have appeared: the animal-fungal and green plant ones. The latter involves also the TOM complexes of algae and protozoans. The amoeba Acanthamoeba castellanii is a free-living non-photosynthetic soil protozoan, whose mitochondria share many bioenergetic properties with mitochondria of plants, animals and fungi. Here, we report that a protein complex, identified electrophysiologically as the A. castellanii TOM complex, contains a homologue of yeast/animal Tom 70. Further, molecular weight of the complex (about 500 kDa) also points to A. castellanii evolutionary relation with fungi and animal. Thus, the data indicates that the TOM complex of A. castellanii is not a typical example of the protozoan TOM complex.
Journal of Bioenergetics 09/2005; 37(4):261-8. · 1.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It is well known that effective exchange of metabolites between mitochondria and the cytoplasm is essential for cell physiology. The key step of the exchange is transport across the mitochondrial outer membrane, which is supported by the voltage-dependent anion-selective channel (VDAC). Therefore, it is clear that the permeability of VDAC must be regulated to adjust its activity to the actual cell needs. VDAC-modulating activities, often referred to as the VDAC modulator, were identified in the intermembrane space of different organism mitochondria but the responsible protein(s) has not been identified as yet. Because the VDAC modulator was reported to act on VDAC of intact mitochondria when added to the cytoplasmic side it has been speculated that a similar modulating activity might be present in the cytoplasm. To check the speculation we used mitochondria of the yeast Saccharomyces cerevisiae as they constitute a perfect model to study VDAC modulation. The mitochondria contain only a single isoform of VDAC and it is possible to obtain viable mutants devoid of the channel (Deltapor1). Moreover, we have recently characterised a VDAC-modulating activity located in the intermembrane space of wild type and Deltapor1 S. cerevisiae mitochondria. Here, we report that the cytoplasm of wild type and Deltapor1 cells of S. cerevisiae contains a VDAC-modulating activity as measured in a reconstituted system and with intact mitochondria. Since quantitative differences were observed between the modulating fractions isolated from wild type and Deltapor1 cells when they were studied with intact wild type mitochondria as well as by protein electrophoresis it might be concluded that VDAC may influence the properties of the involved cytoplasmic proteins. Moreover, the VDAC-modulating activity in the cytoplasm differs distinctly from that reported for the mitochondrial intermembrane space. Nevertheless, both these activities may contribute efficiently to VDAC regulation. Thus, the identification of the proteins is very important.
[Show abstract][Hide abstract] ABSTRACT: The outer mitochondrial membrane of Saccharomyces cerevisiae contains two isoforms of mitochondrial porin, known also as the voltage-dependent anion channel. The isoform termed here porin1 displays channel-forming activity enabling metabolite transport whereas the second one, termed here porin2, does not form a channel and its function is still not clear. We have shown recently that in the absence of porin1, the channel within the protein import machinery (the TOM complex) is essential for metabolite transport across the outer membrane [Kmita and Budzińska, Biochim. Biophys. Acta 1509 (2000) 6044-6050]. Here, we report that the TOM complex channel may also serve as a supplementary pathway for metabolites in the presence of porin1 when the permeability of the latter is limited and the role of the TOM complex seems to increase when porin2 is depleted.
[Show abstract][Hide abstract] ABSTRACT: Thus far, only three channel-forming activities have been identified in the outer membrane of the yeast Saccharomyces cerevisiae mitochondria. Two of them, namely the TOM complex channel (translocase of the outer membrane) and the PSC (peptide-sensitive channel) participate in protein translocation and are probably identical, whereas a channel-forming protein called VDAC (voltage-dependent anion channel) serves as the major pathway for metabolites. The VDAC is present in two isoforms (VDAC1 and VDAC2) of which only VDAC1 has been shown to display channel-forming activity. Moreover, the permeability of VDAC1 has been reported to be limited in uncoupled mitochondria of S. cerevisiae. The presented data indicate that in S. cerevisiae-uncoupled mitochondria, external NADH, applied at higher concentrations (above 50 nmoles per 0.1 mg of mitochondrial protein), may use the TOM complex channel, besides VDAC1, to cross the outer membrane. Thus, the permeability of VDAC1 could be a limiting step in transport of external NADH across the outer membrane and might be supplemented by the TOM complex channel.
Journal of Bioenergetics 05/2001; 33(2):119-26. · 1.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Biochimica et Biophysica Acta 01/2001; 1509(1-2):86-94. · 4.66 Impact Factor