Gruhler A, Ono H, Guiard B, Neupert W, Stuart R A
Institut für Physiologische Chemie, Universität München, Germany.
EMBO J. 1995 Apr 3;14(7):1349-59. doi: 10.1002/j.1460-2075.1995.tb07121.x.
Cytochrome b2 is sorted into the intermembrane space of mitochondria by a bipartite N-terminal targeting and sorting presequence. In an attempt to define the sorting pathway we have identified an as yet unknown import intermediate. Cytochrome b2-dihydrofolate reductase (DHFR) fusion proteins were arrested in the presence of methotrexate (MTX) so that the DHFR domain was at the surface of the outer membrane while the N-terminus reached into the intermembrane space where the sorting signal was removed. This membrane-spanning, mature-sized species was efficiently chased into the mitochondria upon removal of MTX. Thus, an intermediate was generated which was exposed to the intermembrane space but was still associated with the inner membrane. This intermediate was also found upon direct import of cytochrome b2 and derived fusion proteins. These membrane-bound mature-sized cytochrome b2 species loop through the matrix and could be recovered in a complex with mt-Hsp70 and the inner membrane MIM44/ISP45, a component of the inner membrane import apparatus. This novel sorting intermediate can only be explained by a pathway in which cytochrome b2 passes through the matrix. The existence of such an intermediate is inconsistent with a pathway by which entrance of the mature part of cytochrome b2 into the matrix is stopped by the sorting sequence; however, its presence is fully consistent with the conservative sorting pathway.
细胞色素b2通过一个二分的N端靶向和分选前序列被分选到线粒体的膜间隙中。为了确定分选途径，我们鉴定出了一种未知的输入中间体。细胞色素b2-二氢叶酸还原酶（DHFR）融合蛋白在甲氨蝶呤（MTX）存在的情况下被阻滞，使得DHFR结构域位于外膜表面，而N端伸入膜间隙，在那里分选信号被去除。这种跨膜的、成熟大小的物种在去除MTX后能有效地被追踪到线粒体中。因此，产生了一种中间体，它暴露于膜间隙，但仍与内膜相关联。在直接导入细胞色素b2和衍生的融合蛋白时也发现了这种中间体。这些与膜结合的成熟大小的细胞色素b2物种环绕穿过基质，并能与线粒体热休克蛋白70（mt-Hsp70）和内膜导入装置的一个组分——内膜MIM44/ISP45形成复合物而被回收。这种新的分选中间体只能用细胞色素b2穿过基质的途径来解释。这种中间体的存在与细胞色素b2成熟部分进入基质被分选序列阻止的途径不一致；然而，它的存在与保守分选途径完全一致。
