Membrane Potential-Driven Protein Import into Mitochondria

The transport of preproteins into or across the mitochondrial inner membrane requires the membrane potential Δψ across this membrane. Two roles of Δψ in the import of cleavable preproteins have been described: an electrophoretic effect on the positively charged matrix-targeting sequences and the activation of the translocase subunit Tim23. We report the unexpected finding that deletion of a segment within the sorting sequence of cytochrome b(2), which is located behind the matrix-targeting sequence, strongly influenced the Δψ-dependence of import. The differential Δψ-dependence was independent of the submitochondrial destination of the preprotein and was not attributable to the requirement for mitochondrial Hsp70 or Tim23. With a series of preprotein constructs, the net charge of the sorting sequence was altered, but the Δψ-dependence of import was not affected. These results suggested that the sorting sequence contributed to the import driving mechanism in a manner distinct from the two known roles of Δψ. Indeed, a charge-neutral amino acid exchange in the hydrophobic segment of the sorting sequence generated a preprotein with an even better import, i.e. one with lower Δψ-dependence than the wild-type preprotein. The sorting sequence functioned early in the import pathway since it strongly influenced the efficiency of translocation of the matrix-targeting sequence across the inner membrane. These results suggest a model whereby an electrophoretic effect of Δψ on the matrix-targeting sequence is complemented by an import-stimulating activity of the sorting sequence.

将前体蛋白转运至线粒体内膜内或跨线粒体内膜，需要该膜两侧的膜电位Δψ。已有研究阐明了Δψ在可切割前体蛋白导入过程中的两项作用：一是对带正电的基质靶向序列产生电泳效应，二是激活转运酶亚基Tim23。我们在此报告一项意外发现：删除位于基质靶向序列后方的细胞色素b₂分选序列内的一段区域，会显著影响导入过程对Δψ的依赖性。这种Δψ依赖性的差异与前体蛋白的线粒体亚靶位点无关，也并非由线粒体热休克蛋白70（mitochondrial Hsp70）或Tim23的需求所导致。通过一系列前体蛋白构建体，我们改变了分选序列的净电荷，但导入过程对Δψ的依赖性并未受到影响。上述结果表明，分选序列以不同于Δψ已知两项作用的方式，参与了导入驱动机制。实际上，在分选序列的疏水片段中进行电荷中性的氨基酸替换，可得到导入效率更优的前体蛋白——即其对Δψ的依赖性低于野生型前体蛋白。分选序列在导入通路的早期阶段即发挥功能，因其可显著影响基质靶向序列跨线粒体内膜的转位效率。上述结果支持了一种模型：Δψ对基质靶向序列的电泳效应，可由分选序列的导入刺激活性予以补充。