GENETIC ANALYSIS OF PROTEIN TRANSPORT INTO MITOCHONDRIA

酵母属的线粒体醇脱氢酶（ADH III） 酿酒酵母在细胞质中合成并转运到 线粒体 编码这种同工酶ADH 3的核基因已经被发现。 通过利用其与先前克隆的ADH 1的同源性克隆 和ADH 2基因，其编码细胞质同工酶。 完整的 基因的核苷酸序列将通过化学和 酶测序技术。 功能性基因的极限将是 通过S1核酸酶分析确定，以定位5英尺和3英尺末端， 它的mRNA。 线粒体ADH在呼吸和发酵中的功能将被 通过基因破坏来研究。 正常的基因会被 重组DNA技术和酵母转化以及 将研究缺乏功能性ADH 3基因的突变体。 将ADH III转运到线粒体内及其在线粒体内的定位将 通过表征新合成的ADH III多肽来研究。 的 将对推定前体进行鉴定和表征 使用免疫学和蛋白质测序技术。 这些研究将 利用缺乏ADH 1和ADH 2基因的酵母菌株， 也不存在代表ADHI和ADHIII的多肽。 蛋白 将对推定的前体和成熟蛋白进行序列分析， 以鉴定其氨基末端。 ADH III不同区域在线粒体转运中的功能， 加工和靶向正确的线粒体内位点将是 研究了 将在ADH 2和ADH 3之间进行初始融合以研究 氨基末端用于运输的必要性和充分性。 后续 如果氨基末端不被修饰，则在ADH 3和lac Z之间进行融合。 足够了。 将分离在ADH III的线粒体转运中改变的酵母突变体 或者通过选择对烯丙醇的抗性，或者通过选择 对抗霉素的抗性。 ADH III信号序列的突变 和运输蛋白质将被鉴定和表征。 一 还将开发选择或筛选系统， 可能具有多效性转运缺陷的致死突变体。

The mitochondrial alcohol dehydrogenase (ADHIII) of the yeast Saccharomyces cerevisiae is synthesized in the cytoplasm and transported into the mitochondria. The nuclear gene encoding this isozyme, ADH3, has been cloned by taking advantage of its homology to the previously cloned ADH1 and ADH2 genes, which code for the cytoplasmic isozymes. The complete nucleotide sequence of the gene will be determined by chemical and enzymatic sequencing techniques. The limits of the functional gene will be determined by S1 nuclease analysis to locate the 5 feet and 3 feet ends of its mRNA. The function of mitochondrial ADH in respiration and fermentation will be studied by gene disruption. The normal gene will be inactivated by recombinant DNA techniques and yeast transformation and the phenotype of the mutant lacking a functional ADH3 gene will be studied. The transport of ADHIII into and its location within the mitochondria will be studied by characterizing newly-synthesized ADHIII polypeptides. The identification and characterization of a putative precursor will be done using immunological and protein sequencing techniques. These studies will utilize a yeast strain which lacks ADH1 and ADH2 genes so that neither mRNA nor polypeptides representing ADHI and ADHIII are present. Protein sequence analysis of the putative precursor and the mature protein will be performed in order to identify their amino termini. The function of different regions of ADHIII for mitochondrial transport, processing and targetting to the correct intramitochondrial site will be studied. Initial fusions will be made between ADH2 and ADH3 to study the necessity and sufficiency of the amino terminus for transport. Subsequent fusions will be made between ADH3 and lac Z if the amino terminus is not sufficient. Yeast mutants altered in mitochondrial transport of ADHIII will be isolated either by selection for resistance to allyl alcohol or by selection for resistance to antimycin. Mutants altered in the signal sequence of ADHIII and in transport proteins will be identified and characterized. A selection or screening system will also be developed to isolate conditional lethal mutants which might have pleiotropic transport defects.