INTRACELLULAR TARGETING AND IMPORT OF AN F1-ATPASE BETA-SUBUNIT-BETA-GALACTOSIDASE HYBRID PROTEIN INTO YEAST MITOCHONDRIA
INTRACELLULAR TARGETING AND IMPORT OF AN F1-ATPASE BETA-SUBUNIT-BETA-GALACTOSIDASE HYBRID PROTEIN INTO YEAST MITOCHONDRIA
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DOI：
10.1073/pnas.81.13.3983
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发表时间：
1984-01-01
期刊：
影响因子：
--
通讯作者：
EMR, SD
中科院分区：
文献类型：
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作者：
DOUGLAS, MG;GELLER, BL;EMR, SD
编码酵母线粒体F1 ATP酶β的基因亚基（ATP 2）与大肠杆菌lacZ基因融合。嵌合ATP 2-lacZ基因编码由F1-ATP酶β的约350个氨基酸组成的杂合蛋白。在其氨基末端的β-亚基和lacZ基因产物的大的酶活性部分，半乳糖苷酶（β- D-半乳糖水解酶，EC www.example.com）。β-亚基-β-半乳糖苷酶杂合蛋白在E.大肠杆菌和酵母菌。在酵母中，这种杂合分子靶向于线粒体，并且在其中外膜酶标记被消化的条件下，在分离的线粒体中被保护免于添加蛋白酶。在质粒p β Z1上携带ATP 2-lacZ基因融合体的酵母细胞不能在不可发酵的C源上生长。当p β Z1质粒丢失时，不可发酵底物的治愈宿主菌株的生长恢复。在β-亚基-β-半乳糖苷酶杂合蛋白，线粒体膜的能量转导能力，如通过32 Pi-ATP交换反应测量的，仅为在不存在基因融合产物的情况下测量的9%。它是β的存在。亚基-β-线粒体内干扰呼吸生长所必需的功能的半乳糖苷酶杂合蛋白。这些观察结果开辟了负责线粒体蛋白质递送的信号和细胞机制的遗传表征的前景。
The gene coding for the yeast mitochondrial F1ATPase .beta. subunit (ATP2) was fused to the Escherichia coli lacZ gene. The chimeric ATP2-lacZ gene codes for a hybrid protein consisting of some 350 amino acids of the F1-ATPase .beta. subunit at its amino terminus and a large enzymatically active portion of the lacZ gene product, .beta.-galactosidase (.beta.-D-galactohydrolase, EC 3.2.1.23), at its carboxyl terminus. The .beta.-subunit-.beta.-galactosidase hybrid protein is expressed in both E. coli and yeast. In yeast, this hybrid molecule is targeted to the mitochondrion and is protected in isolated mitochondria from added protease under conditions in which an outer membrane enzymatic marker is digested. Yeast cells carrying the ATP2-lacZ gene fusion on plasmid p.beta.Z1 are unable to grow on a nonfermentable C source. Upon loss of the p.beta.Z1 plasmid, growth of the cured host strain of the nonfermentable substrate is restored. In the presence of the .beta.-subunit-.beta.-galactosidase hybrid protein, the energy-transducing capacity of the mitochondrial membrane as measured by the 32Pi-ATP exchange reaction is only 9% of that measured in the absence of the gene fusion product. It is the presence of the .beta.-subunit-.beta.-galactosidase hybrid protein within mitochondria that interferes with function(s) essential for respiratory growth. These observations open up the prospect of genetic characterization of the signals and cellular machinery responsible for mitochondrial protein delivery.