Genetic Engineering Laboratory, Molecular and Human Genetics Division, CSIR-Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Calcutta 700 032, India.
Biochem Biophys Res Commun. 2013 Sep 13;439(1):23-9. doi: 10.1016/j.bbrc.2013.08.036. Epub 2013 Aug 17.
Very little is known about how nucleic acids are translocated across membranes. The multi-subunit RNA Import Complex (RIC) from mitochondria of the kinetoplastid protozoon Leishmania tropica induces translocation of tRNAs across artificial or natural membranes, but the nature of the translocation pore remains unknown. We show that subunits RIC6 and RIC9 assemble on the membrane in presence of subunit RIC4A to form complex R3. Atomic Force Microscopy of R3 revealed particles with an asymmetric surface groove of ∼20 nm rim diameter and ∼1 nm depth. R3 induced translocation of tRNA into liposomes when the pH of the medium was lowered to ∼6 in the absence of ATP. R3-mediated tRNA translocation could also be induced at neutral pH by a K(+) diffusion potential with an optimum of 60-70 mV. Point mutations in the Cys2-His2 Fe-binding motif of RIC6, which is homologous to the respiratory Complex III Fe-S protein, abrogated import induced by low pH but not by K(+) diffusion potential. These results indicate that the R3 complex forms a pore that is gated by a proton-generated membrane potential and that the Fe-S binding region of RIC6 has a role in proton translocation. The tRNA import complex of L. tropica thus contains a novel macromolecular channel distinct from the mitochondrial protein import pore that is apparently involved in tRNA import in some species.
关于核酸如何穿过膜的机制知之甚少。来自锥虫热带利什曼原虫线粒体的多亚基 RNA 导入复合物（RIC）可诱导 tRNA 穿过人工或天然膜的易位，但易位孔的性质仍不清楚。我们发现，在存在 RIC4A 亚基的情况下，亚基 RIC6 和 RIC9 会在膜上组装形成 R3 复合物。原子力显微镜观察 R3 发现，其表面具有不对称的 20nm 边缘直径和 1nm 深度的环形凹槽。当介质的 pH 在没有 ATP 的情况下降低到约 6 时，R3 诱导 tRNA 进入脂质体的易位。在中性 pH 下，通过 K+扩散电势也可以诱导 R3 介导的 tRNA 易位，最佳电势为 60-70 mV。RIC6 的 Cys2-His2 Fe 结合基序中的点突变（与呼吸复合物 III Fe-S 蛋白同源）消除了由低 pH 诱导的导入，但没有消除由 K+扩散电势诱导的导入。这些结果表明，R3 复合物形成了一个由质子产生的膜电位控制的孔，并且 RIC6 的 Fe-S 结合区域在质子易位中起作用。因此，热带利什曼原虫的 tRNA 导入复合物包含一种不同于线粒体蛋白导入孔的新型大分子通道，该通道显然参与了某些物种的 tRNA 导入。
