Kmita H, Budzińska M
Department of Bioenergetics, Institute of Molecular Biology and Biotechnology, Poznan University, Fredry 10, 61-701, Poznan, Poland.
Biochim Biophys Acta. 2000 Dec 20;1509(1-2):86-94. doi: 10.1016/s0005-2736(00)00284-4.
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.
负责代谢物通过酿酒酵母线粒体（线粒体孔蛋白（也称为电压依赖性阴离子选择性通道）缺失，此处称为孔蛋白1）外膜进行转运的蛋白质仍未确定。据推测，这种转运可能由外膜的蛋白质导入机制即TOM复合体（外膜转位酶）提供支持。我们在此证明，在缺乏功能性孔蛋白1的情况下，被称为pb(2)-DHFR的融合蛋白（由酵母细胞色素b(2)前体蛋白的前167个氨基酸与小鼠二氢叶酸还原酶连接而成）对TOM复合体的阻断限制了外部NADH进入线粒体。这是通过该阻断抑制内膜外表面的适当脱氢酶对外部NADH氧化的能力来测定的。这种抑制作用取决于外部NADH的浓度，并随着底物量的减少而增加。当每50微克线粒体蛋白中存在1微克pb(2)-DHFR时，以每毫克线粒体蛋白70纳摩尔的浓度加入外部NADH时，几乎观察到了定量抑制。另一方面，外部NADH以浓度依赖性方式降低孔蛋白1缺失的线粒体中TOM复合体转位位点处pb(2)-DHFR的结合水平。我们的数据确定了TOM复合体在外部NADH跨孔蛋白1缺失的线粒体外膜转运中的重要作用。
