Involvement of the TOM complex in external NADH transport into yeast mitochondria depleted of mitochondrial porin1.
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.
Kmita
H
H
Department of Bioenergetics, Institute of Molecular Biology and Biotechnology, Poznan University, Fredry 10, 61-701, Poznan, Poland. kmita@main.amu.edu.pl
Budzińska
M
M
D001692:Biological Transport; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008928:Mitochondria / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D009243:NAD / Q000378:metabolism* / Q000494:pharmacology; D010084:Oxidation-Reduction; D018272:Porins / Q000172:deficiency*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D050994:Voltage-Dependent Anion Channels
D005656:Fungal Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D018272:Porins; D029701:Saccharomyces cerevisiae Proteins; C489039:TOM70 protein, S cerevisiae; C066555:Tom40 protein, S cerevisiae; D050994:Voltage-Dependent Anion Channels; D009243:NAD