HTS1 encodes both the cytoplasmic and mitochondrial histidyl-tRNA synthetase of Saccharomyces cerevisiae: mutations alter the specificity of compartmentation.
HTS1 encodes both the cytoplasmic and mitochondrial histidyl-tRNA synthetase of Saccharomyces cerevisiae: mutations alter the specificity of compartmentation.
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HTS1 编码酿酒酵母的细胞质和线粒体组氨酰-tRNA 合成酶：突变改变了区室划分的特异性。
DOI：
10.1093/genetics/132.4.987
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发表时间：
1992
期刊：
影响因子：
3.3
通讯作者：
Fink,GR
中科院分区：
文献类型：
--
作者：
Chiu,MI;Mason,TL;Fink,GR
遗传和生化证据表明，一个核基因HTS 1编码线粒体和细胞质组氨酰-tRNA合成酶（Hts）。该基因指定两条消息，一条具有两个帧内ATG（-60和+1），另一条仅具有下游ATG（+1）。我们已经做了一组新的突变，使我们能够只表达线粒体或细胞质形式，并比较了这些突变体和野生型的Hts 1蛋白的亚细胞分布，使用与线粒体和细胞质Hts 1以及Hts 1：：LacZ融合相互作用的抗体。上游ATG（-60）突变或前序列移码突变仅影响线粒体酶，而不影响细胞质酶。下游ATG的突变（+1 ATG至ATC）破坏胞质酶的功能，但不影响线粒体酶的功能。该构建体的过表达恢复细胞质功能。表达含有前20个氨基末端残基缺失的截短形式的Hts（Htsc）的细胞产生功能性胞质酶，其不提供线粒体功能。这种截短的细胞质蛋白的过表达提供线粒体功能，并在线粒体中产生可检测水平的合成酶。这些实验表明，Hts 1包含两个域，一起允许有效的本地化Htsm的线粒体前体的氨基末端前序列，可能会被切割后交付的线粒体和第二个氨基末端序列（残基21-53）存在于前体和细胞质的形式。两者本身都不足以充当有效的线粒体靶向信号。使用我们的抗体，我们已经能够检测到分子量增加的蛋白质，其对应于预测的前体。总之，这些研究表明，Hts蛋白的区室化的特异性取决于一级序列和细胞中蛋白质的浓度。
Genetic and biochemical evidence shows that a single nuclear gene HTS1 encodes both the mitochondrial and cytoplasmic histidyl-tRNA synthetases (Hts). The gene specifies two messages, one with two in-frame ATGs (-60 and +1) and another with only the downstream ATG (+1). We have made a new set of mutations that enables us to express only the mitochondrial or the cytoplasmic form and compared the subcellular distribution of the Hts1 protein in these mutants and wild type, using an antibody that interacts with both the mitochondrial and cytoplasmic Hts1 as well as Hts1::LacZ fusions. Mutations in the upstream ATG (-60) or frameshift mutations in the presequence affect only the mitochondrial enzyme and not the cytoplasmic enzyme. Mutations in the downstream ATG (+1 ATG to ATC) destroy the function of the cytosolic enzyme, but do not affect the function of the mitochondrial enzyme. Overexpression of this construct restores cytoplasmic function. Cells expressing a truncated form of Hts containing a deletion of the first 20 amino-terminal residues (Htsc) produce a functional cytoplasmic enzyme, which does not provide mitochondrial function. Overexpression of this truncated cytoplasmic protein provides mitochondrial function and produces detectable levels of the synthetase in the mitochondrion. These experiments suggest that Hts1 contains two domains that together allow efficient localization of Htsm to the mitochondrion: an amino-terminal presequence in the mitochondrial precursor that is likely cleaved upon delivery to the mitochondrion and a second amino-terminal sequence (residues 21-53) present in both the precursor and the cytoplasmic form. Neither one by itself is sufficient to act as an efficient mitochondrial targeting signal. Using our antibody we have been able to detect a protein of increased molecular mass that corresponds to that of the predicted precursor. Taken together these studies show that the specificity of compartmentation of the Hts protein depends upon both the primary sequence and the concentration of the protein in the cell.