Esaki M, Kanamori T, Nishikawa S i, Endo T
Department of Chemistry, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.
Proc Natl Acad Sci U S A. 1999 Oct 12;96(21):11770-5. doi: 10.1073/pnas.96.21.11770.
The import of cytochrome b(2) into mitochondria consists of two steps. The translocation of the first part of the presequence across the inner membrane is coupled with the translocation of the tightly folded heme-binding domain across the outer membrane and requires a membrane potential DeltaPsi and the functions of mitochondrial Hsp70 (mHsp70) in the matrix. Once the heme-binding domain has passed the outer membrane, the translocation of the rest of the polypeptide chain across the outer membrane becomes independent of DeltaPsi and mHsp70. Here we analyzed the late DeltaPsi- and mHsp70-independent step in the transport of cytochrome b(2) fusion proteins into the intermembrane space (IMS). The import of the cytochrome b(2) fusion proteins containing two protein domains linked by a spacer segment into mitochondria was arrested at a stage at which one domain folded on each side of the outer membrane, along the pathway that is consistent with the stop-transfer model. The mature-size form of the translocation intermediate could move across the outer membrane in both directions, and the stabilization of the protein domain in the IMS promoted the forward translocation. On the other hand, the intermediate-size form of the translocation intermediate, which retains the anchorage to the inner membrane, was transported into the IMS independently of the stability of the protein domain in the IMS. These results suggest that two distinct mechanisms, the Brownian ratchet and the anchor diffusion mechanisms, can operate for the transmembrane movement of the mature-size form and the intermediate-size form, respectively, of cytochrome b(2) species.
细胞色素b(2)导入线粒体包括两个步骤。前导序列第一部分穿过内膜的转运与紧密折叠的血红素结合结构域穿过外膜的转运相偶联，并且需要膜电位ΔΨ以及基质中线粒体Hsp70（mHsp70）的功能。一旦血红素结合结构域穿过外膜，多肽链其余部分穿过外膜的转运就变得独立于ΔΨ和mHsp70。在这里，我们分析了细胞色素b(2)融合蛋白转运到膜间隙（IMS）过程中晚期不依赖于ΔΨ和mHsp70的步骤。含有由间隔片段连接的两个蛋白质结构域的细胞色素b(2)融合蛋白导入线粒体的过程在一个阶段被阻断，在这个阶段，一个结构域在外膜两侧折叠，沿着与停止转运模型一致的途径。转运中间体的成熟大小形式可以双向穿过外膜，并且蛋白质结构域在IMS中的稳定促进了向前转运。另一方面，保留与内膜锚定的转运中间体的中间大小形式被独立于其在IMS中蛋白质结构域的稳定性转运到IMS中。这些结果表明，布朗棘轮和锚定扩散机制这两种不同的机制可分别作用于细胞色素b(2)物种的成熟大小形式和中间大小形式的跨膜移动。
