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.
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.
Edited by Vladimir Skulachev
Keywords:Copper-and zinc-containing superoxide dismutase Voltage dependent anion selective channel Channel activity Tob55/Sam50 Tom40 Saccharomyces cerevisiae a b s t r a c t Available data suggest that a copper-and zinc-containing dismutase (CuZnSOD) plays a significant role in protecting eukaryotic cells against oxidative modifications which may contribute to cell aging. Here we demonstrated that depletion of CuZnSOD in Saccharomyces cerevisiae cells (Dsod1 cells) affected distinctly channel activity of VDAC (voltage dependent anion selective channel) and resulted in a moderate reduction in VDAC levels as well as in levels of protein crucial for VDAC import into mitochondria, namely Tob55/Sam50 and Tom40. The observed alterations may result in mitochondriopathy and subsequently in the shortening of the replicative life span observed for S. cerevisiae Dsod1 cells.
Voltage dependent anion channel (VDAC) was identified in 1976 and since that time has been extensively studied. It is well known that VDAC transports metabolites across the outer mitochondrial membrane. The simple transport function is indispensable for proper mitochondria functions and, consequently for cell activity, and makes VDAC crucial for a range of cellular processes including ATP rationing, Ca2+ homeostasis and apoptosis execution. Here, we review recent data obtained for Saccharomyces cerevisiae cells used as a model system concerning the putative role of VDAC in communication between mitochondria and the nucleus. The S. cerevisiae VDAC isoform known as VDAC1 (termed here YVDAC) mediates the cytosol reduction/oxidation (redox) state that contributes to regulation of expression and activity of cellular proteins including proteins that participate in protein import into mitochondria and antioxidant enzymes. Simultaneously, copper-and-zinc-containing superoxide dismutase (CuZnSOD) plays an important role in controlling YVDAC activity and expression levels. Thus, it is proposed that VDAC constitutes an important component of a regulatory mechanism based on the cytosol redox state.
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