Molecular mechanisms of protein sorting between the mitochondrial outer and inner membranes

在细胞质中合成的线粒体蛋白质被运输并定位于线粒体的特定区室。以70 kDa的外膜蛋白和细胞色素c_1为酵母细胞的代表蛋白，研究了外膜和内膜之间的线粒体内分选。细胞色素c_1的前序列，其功能作为一个信号的内膜被取代的70 kDa蛋白的N-末端区域的各种长度。N端61个残基的融合蛋白（61mC_1）定位于外膜，N端29个残基以下的融合蛋白定位于内膜。后者的融合蛋白被证明组装成琥珀酸-细胞色素c还原酶，而没有蛋白水解去除融合区，并恢复细胞色素c_1缺陷突变酵母（MH 6 -16）的呼吸。这些数据表明，外膜蛋白的靶向和锚定信号将内膜蛋白定位于外膜，只有靶序列将其引导至内膜。用编码61mC_1的质粒转化的细胞色素c_1缺陷型酵母MH 6 -16不能在非发酵培养基上生长（甘油^-），这是由于该蛋白质位于外膜。对在甘油上恢复生长的突变的选择被应用于转化体，并在宿主基因组中产生一类邀请。我们现在将这种突变定性为线粒体蛋白分选突变体，目前分离出了参与恢复的DNA片段。

Mitochondrial proteins synthesized in the cytoplasm are transported and localized to a specific compartment of the mitochondrion. The intramitochondrial sorting between the outer and inner membranes was investigated using a 70 kDa outer membrane protein and cytochrome c_1 as representative proteins in yeast cells. The presequence of cytochrome c_1, which functions as a signal to the inner membrane was replaced by various lengths of the N-terminal region of the 70 kDa protein. The fused protein with the N-terminal 61 residues(61mC_1) was localized to the outer membrane and those with less than 29 residues to the inner membrane. The latter fusion proteins were shown to be assembled into succinate-eytochrome c reductase without proteolytic removal of the fused region and restore the respiration of a cytochrome c_1 deficient mutant yeast(MH6-16). These date indicate that targeting and anchoring signals of the outer membrane protein localize the inner membrane protein to the outer membrane and that the target sequence alone directs it to the inner membrane.The cytochrome c_1 deficient mutant yeast, MH6-16 transformed with a plasmid encoding 61mC_1 was not able to grow on a non- fermentable medium(glycerol^-), due to location of this protein to the outer membrane. A selection for mutations which restored growth on glycerol was applied to the transformants and yield a class of invitation in the host genome. We are now characterizing this mutation as a mitochondrial protein sorting mutant and a DNA fragment involved in the restoration are currently isolated.