Kanamori T, Nishikawa S, Nakai M, Shin I, Schultz P G, Endo T
Department of Chemistry, Faculty of Science, Nagoya University, Nagoya 464-8602, Japan.
Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):3634-9. doi: 10.1073/pnas.96.7.3634.
Translocation of mitochondrial precursor proteins across the mitochondrial outer membrane is facilitated by the translocase of the outer membrane (TOM) complex. By using site-specific photocrosslinking, we have mapped interactions between TOM proteins and a mitochondrial precursor protein arrested at two distinct stages, stage A (accumulated at 0 degrees C) and stage B (accumulated at 30 degrees C), in the translocation across the outer membrane at high resolution not achieved previously. Although the stage A and stage B intermediates were assigned previously to the forms bound to the cis site and the trans site of the TOM complex, respectively, the results of crosslinking indicate that the presequence of the intermediates at both stage A and stage B is already on the trans side of the outer membrane. The mature domain is unfolded and bound to Tom40 at stage B whereas it remains folded at stage A. After dissociation from the TOM complex, translocation of the stage B intermediate, but not of the stage A intermediate, across the inner membrane was promoted by the intermembrane-space domain of Tom22. We propose a new model for protein translocation across the outer membrane, where translocation of the presequence and unfolding of the mature domain are not necessarily coupled.
线粒体外膜转位酶（TOM）复合体促进线粒体前体蛋白穿过线粒体外膜。通过使用位点特异性光交联技术，我们在高分辨率下绘制了TOM蛋白与处于两个不同阶段（阶段A：在0℃积累；阶段B：在30℃积累）的线粒体前体蛋白之间的相互作用图谱，这是之前未达到的高分辨率，该前体蛋白处于穿过外膜的转位过程中。尽管阶段A和阶段B中间体之前分别被认为是与TOM复合体的顺式位点和反式位点结合的形式，但交联结果表明，阶段A和阶段B中间体的前导序列都已经在外膜的反式侧。成熟结构域在阶段B时展开并与Tom40结合，而在阶段A时它保持折叠状态。从TOM复合体解离后，Tom22的膜间隙结构域促进了阶段B中间体而非阶段A中间体穿过内膜的转位。我们提出了一种新的蛋白穿过外膜的模型，其中前导序列的转位和成熟结构域的展开不一定是偶联的。
