Rapaport D, Mayer A, Neupert W, Lill R
Institut für Physiologische Chemie, Physikalische Biochemie und Zellbiologie der Universität München, Goethestrabetae 33, 80336 München, Federal Republic of Germany.
J Biol Chem. 1998 Apr 10;273(15):8806-13. doi: 10.1074/jbc.273.15.8806.
Translocation of preproteins across the mitochondrial outer membrane is mediated by the TOM complex. Our previous studies led to the concept of two preprotein binding sites acting in series, the surface-exposed cis site and the trans site exposed to the intermembrane space. We report here that preproteins are bound to the cis site in a labile fashion even at low ionic strength, whereas intermediates arrested at the trans site remained firmly bound at higher salt concentration. The stability of the trans site intermediate results from interactions of both the presequence and unfolded parts of the mature part of the preprotein with the TOM complex. Binding to the trans site proceeded at rates comparable with those of unfolding of the mature domain and appeared to be kinetically limited by the unfolding reaction. Efficient binding to the trans site and unfolding were observed with both outer membrane vesicles and intact mitochondria whose membrane potential, DeltaPsi, was dissipated. Upon re-establishing DeltaPsi, trans site-bound preprotein resumed translocation into the matrix. The rates of unfolding and binding to the trans site were the same as those for translocation into intact energized mitochondria. We conclude that preprotein unfolding in intact mitochondria can take place without the involvement of the translocation machinery of the inner membrane and, in particular, the matrix Hsp70 chaperone. Further, preprotein unfolding at the outer membrane can be a rate-limiting step for formation of the trans site intermediate and for the entire translocation reaction.
前体蛋白穿过线粒体外膜的转运由TOM复合体介导。我们之前的研究提出了两个串联作用的前体蛋白结合位点的概念，即表面暴露的顺式位点和暴露于膜间隙的反式位点。我们在此报告，即使在低离子强度下，前体蛋白也以不稳定的方式结合到顺式位点，而停滞在反式位点的中间体在较高盐浓度下仍保持牢固结合。反式位点中间体的稳定性源于前体蛋白前导序列和成熟部分未折叠部分与TOM复合体的相互作用。与反式位点的结合速率与成熟结构域的解折叠速率相当，并且在动力学上似乎受解折叠反应的限制。在膜电位ΔΨ消散的外膜囊泡和完整线粒体中均观察到与反式位点的有效结合和解折叠。重新建立ΔΨ后，反式位点结合的前体蛋白恢复向基质的转运。解折叠和与反式位点结合的速率与向完整的有活性线粒体转运的速率相同。我们得出结论，完整线粒体中的前体蛋白解折叠可以在不涉及内膜转运机制特别是基质Hsp70伴侣的情况下发生。此外，外膜处的前体蛋白解折叠可能是反式位点中间体形成和整个转运反应的限速步骤。
