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首页> 外文期刊>Molecular and Cellular Biology >Primary structure requirements for correct sorting of the yeast mitochondrial protein ADH III to the yeast mitochondrial matrix space.
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Primary structure requirements for correct sorting of the yeast mitochondrial protein ADH III to the yeast mitochondrial matrix space.

机译：正确分类酵母线粒体蛋白ADH III到酵母线粒体基质空间的基本结构要求。

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摘要

Alcohol dehydrogenase isoenzyme III (ADH III) in Saccharomyces cerevisiae, the product of the ADH3 gene, is located in the mitochondrial matrix. The ADH III protein was synthesized as a larger precursor in vitro when the gene was transcribed with the SP6 promoter and translated with a reticulocyte lysate. A precursor of the same size was detected when radioactively pulse-labeled proteins were immunoprecipitated with anti-ADH antibody. This precursor was rapidly processed to the mature form in vivo with a half-time of less than 3 min. The processing was blocked if the mitochondria were uncoupled with carbonyl cyanide m-chlorophenylhydrazone. Mutant enzymes in which only the amino-terminal 14 or 16 amino acids of the presequence were retained were correctly targeted and imported into the matrix. A mutant enzyme that was missing the amino-terminal 17 amino acids of the presequence produced an active enzyme, but the majority of the enzyme activity remained in the cytoplasmic compartment on cellular fractionation. Random amino acid changes were produced in the wild-type presequence by bisulfite mutagenesis of the ADH3 gene. The resulting ADH III protein was targeted to the mitochondria and imported into the matrix in all of the mutants tested, as judged by enzyme activity. Mutants containing amino acid changes in the carboxyl-proximal half of the ADH3 presequence were imported and processed to the mature form at a slower rate than the wild type, as judged by pulse-chase studies in vivo. The unprocessed precursor appeared to be unstable in vivo. It was concluded that only a small portion of the presequence contains the necessary information for correct targeting and import. Furthermore, the information for correct proteolytic processing of the presequence appears to be distinct from the targeting information and may involve secondary structure information in the presequence.
机译：ADH3基因的产物啤酒酵母中的酒精脱氢酶同工酶III（ADH III）位于线粒体基质中。当用SP6启动子转录该基因并用网织红细胞裂解液翻译时，ADH III蛋白在体外合成为较大的前体。用抗ADH抗体免疫沉淀放射性脉冲标记的蛋白后，可以检测到大小相同的前体。该前体在不到三分钟的时间内便在体内迅速加工成成熟形式。如果线粒体未与羰基氰化物间氯苯hydr偶合，则该过程将被阻止。正确保留了仅保留该序列的氨基末端14或16个氨基酸的突变酶，并将其导入基质中。缺少该序列的氨基末端17个氨基酸的突变酶产生了一种活性酶，但大多数酶活性保留在细胞分级分离的细胞质区室中。通过ADH3基因的亚硫酸氢盐诱变，在野生型序列中产生随机的氨基酸变化。根据酶活性判断，所得的ADH ​​III蛋白靶向线粒体，并导入所有测试突变体的基质中。根据体内脉冲追踪研究的判断，ADH3前序羧基近半部分中含有氨基酸变化的突变体以比野生型慢的速率输入并加工成成熟形式。未经加工的前体在体内似乎不稳定。得出的结论是，只有一小部分的预序列包含正确定位和导入所需的信息。此外，用于序列的正确蛋白水解处理的信息似乎与靶向信息不同，并且可能在序列中涉及二级结构信息。

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