Krause-Buchholz U, Tzschoppe K, Paret C, Ostermann K, Rödel G
Institute of Genetics, Dresden University of Technology, D-01062 Dresden, Germany.
Yeast. 2000 Mar 15;16(4):353-63.
Translation of cytochrome b mRNA in yeast mitochondria requires activation by the nuclear-encoded Cbs1p. According to the current model, Cbs1p tethers cytochrome b mRNA to the inner mitochondrial membrane via interaction with the 5'-untranslated leader. Cbs1p is predicted to be a hydrophilic protein with two hydrophobic segments near the carboxyl-terminal end, which are both too short to span the membrane. Nevertheless Cbs1p is tightly associated with the mitochondrial membrane, as shown by its behaviour in extraction experiments with taurodeoxycholate. In an attempt to define functionally important regions of Cbs1p, we created a number of mutant alleles by random and directed mutagenesis. We report that a Cbs1p mutant protein lacking the mitochondrial presequence is still able to complement a Deltacbs1 strain, suggesting that the presequence does not contain essential mitochondrial targeting information. Mutations in a cluster of positively charged amino acids at the extremeC-terminus have no effect on Cbs1p function, but removal of this segment severely impairs Cbs1p function. Truncation of 12 or more amino acids from the C-terminus results in a completely defective protein. We further show that both short hydrophobic regions are essential for Cbs1p function, although membrane association is observed even in the absence of these regions.
酵母线粒体中细胞色素b mRNA的翻译需要由核编码的Cbs1p激活。根据目前的模型，Cbs1p通过与5'-非翻译前导序列相互作用，将细胞色素b mRNA tether在线粒体内膜上。预测Cbs1p是一种亲水蛋白，在羧基末端附近有两个疏水片段，这两个片段都太短而无法跨越膜。然而，正如其在牛磺脱氧胆酸盐提取实验中的行为所示，Cbs1p与线粒体膜紧密相关。为了确定Cbs1p功能上重要的区域，我们通过随机和定向诱变创建了许多突变等位基因。我们报告说，缺乏线粒体前导序列的Cbs1p突变蛋白仍然能够互补Deltacbs1菌株，这表明前导序列不包含必需的线粒体靶向信息。极端C末端的一组带正电荷氨基酸的突变对Cbs1p功能没有影响，但去除该片段会严重损害Cbs1p功能。从C末端截短12个或更多氨基酸会导致完全有缺陷的蛋白质。我们进一步表明，尽管即使在没有这些区域的情况下也能观察到膜结合，但两个短疏水区域对Cbs1p功能都是必不可少的。
