Bedwell D M, Strobel S A, Yun K, Jongeward G D, Emr S D
Division of Biology, California Institute of Technology, Pasadena 91125.
Mol Cell Biol. 1989 Mar;9(3):1014-25. doi: 10.1128/mcb.9.3.1014-1025.1989.
The Saccharomyces cerevisiae F1-ATPase beta subunit precursor contains redundant mitochondrial protein import information at its NH2 terminus (D. M. Bedwell, D. J. Klionsky, and S. D. Emr, Mol. Cell. Biol. 7:4038-4047, 1987). To define the critical sequence and structural features contained within this topogenic signal, one of the redundant regions (representing a minimal targeting sequence) was subjected to saturation cassette mutagenesis. Each of 97 different mutant oligonucleotide isolates containing single (32 isolates), double (45 isolates), or triple (20 isolates) point mutations was inserted in front of a beta-subunit gene lacking the coding sequence for its normal import signal (codons 1 through 34 were deleted). The phenotypic and biochemical consequences of these mutations were then evaluated in a yeast strain deleted for its normal beta-subunit gene (delta atp2). Consistent with the lack of an obvious consensus sequence for mitochondrial protein import signals, many mutations occurring throughout the minimal targeting sequence did not significantly affect its import competence. However, some mutations did result in severe import defects. In these mutants, beta-subunit precursor accumulated in the cytoplasm, and the yeast cells exhibited a respiration defective phenotype. Although point mutations have previously been identified that block mitochondrial protein import in vitro, a subset of the mutations reported here represents the first single missense mutations that have been demonstrated to significantly block mitochondrial protein import in vivo. The previous lack of such mutations in the beta-subunit precursor apparently relates to the presence of redundant import information in this import signal. Together, our mutants define a set of constraints that appear to be critical for normal activity of this (and possibly other) import signals. These include the following: (i) mutant signals that exhibit a hydrophobic moment greater than 5.5 for the predicted amphiphilic alpha-helical conformation of this sequence direct near normal levels of beta-subunit import (ii) at least two basic residues are necessary for efficient signal function, (iii) acidic amino acids actively interfere with import competence, and (iv) helix-destabilizing residues also interfere with signal function. These experimental observations provide support for mitochondrial protein import models in which both the structure and charge of the import signal play a critical role in directing mitochondrial protein targeting and import.
酿酒酵母F1 - ATP合酶β亚基前体在其NH2末端含有冗余的线粒体蛋白导入信息（D.M.贝德韦尔、D.J.克利昂斯基和S.D.埃姆尔，《分子细胞生物学》7:4038 - 4047，1987年）。为了确定这个拓扑信号中包含的关键序列和结构特征，对其中一个冗余区域（代表一个最小靶向序列）进行了饱和盒式诱变。将97个不同的含有单（32个分离株）、双（45个分离株）或三（20个分离株）点突变的突变寡核苷酸分离株分别插入到一个缺失其正常导入信号编码序列（第1至34个密码子被删除）的β亚基基因前面。然后在一个缺失其正常β亚基基因（δatp2）的酵母菌株中评估这些突变的表型和生化后果。与线粒体蛋白导入信号缺乏明显的共有序列一致，在整个最小靶向序列中发生的许多突变并没有显著影响其导入能力。然而，一些突变确实导致了严重的导入缺陷。在这些突变体中，β亚基前体积聚在细胞质中，酵母细胞表现出呼吸缺陷表型。虽然以前已经鉴定出在体外阻断线粒体蛋白导入的点突变，但这里报道的一部分突变代表了首次被证明在体内显著阻断线粒体蛋白导入的单个错义突变。以前在β亚基前体中缺乏此类突变显然与该导入信号中存在冗余导入信息有关。总之，我们的突变体定义了一组似乎对这个（可能还有其他）导入信号的正常活性至关重要的限制条件。这些条件包括：（i）对于该序列预测的两亲性α螺旋构象，疏水矩大于5.5的突变信号指导β亚基接近正常水平的导入；（ii）高效信号功能至少需要两个碱性残基；（iii）酸性氨基酸会积极干扰导入能力；（iv）破坏螺旋的残基也会干扰信号功能。这些实验观察结果为线粒体蛋白导入模型提供了支持，在该模型中，导入信号的结构和电荷在指导线粒体蛋白靶向和导入中都起着关键作用。
