Mitochondrial translocation and processing of the precursor to the alpha-subunit of rat liver succinyl-CoA synthetase.
Mitochondrial translocation and processing of the precursor to the alpha-subunit of rat liver succinyl-CoA synthetase.
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线粒体易位和大鼠肝脏琥珀酰辅酶A合成酶α亚基前体的加工。
DOI：
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发表时间：
1990
期刊：
影响因子：
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通讯作者：
W. A. Bridger
中科院分区：
文献类型：
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作者：
R. Majumdar;W. A. Bridger
琥珀酰辅酶A合成酶在线粒体基质中以α-β-二聚体的形式发挥作用。因此，它的组成亚基有望在细胞核中编码，并在细胞质中合成，作为包含线粒体易位信号序列的前体。我们之前已经报道了一个大鼠肝脏cdna克隆(Lambda SCS19)的分离和测序，该克隆明显编码α-亚基的细胞质前体。在这里，我们报道了该cDNA插入片段的mRNA转录本的制备及其体外翻译，以产生标记蛋白，该标记蛋白可以跨随后添加的大鼠肝脏线粒体膜转运。转位伴随着蛋白质降解过程，将34.5千道尔顿的前体转化为32千道尔顿的成熟亚基形式。用顺序Edman降解法测定了GTP特异性同工酶成熟α-亚基的N-末端序列，并与由该基因推导的氨基酸序列进行了比较。这证实了克隆的序列编码GTP特异性的α-亚基，并确定了裂解点在组氨酸和甘氨酰残基之间，信号序列由27个残基组成。该信号序列与已阐明的其他线粒体靶向序列(主要是酵母线粒体前体)具有相同的特征，包括形成两亲性螺旋的可能性。进口依赖于ATP的存在，并被降低线粒体膜电位的化合物抑制。前体的易位对于网织红细胞翻译系统产生的前体是有效的，但对于由小麦胚芽提取物翻译的产物则看不到，这表明后者可能缺乏靶向和进口过程所必需的因子或成分。
Succinyl-CoA synthetase functions in the mitochondrial matrix as an alpha beta-dimer. Its constitutive subunits are thus expected to be encoded in the nucleus and synthesized in the cytoplasm as precursors containing signal sequences for mitochondrial translocation. We have previously reported the isolation and sequence of a rat liver cDNA clone (lambda SCS19) that apparently encodes the cytoplasmic precursor to the alpha-subunit. Here we report the preparation of mRNA transcripts of this cDNA insert and their in vitro translation to produce labeled protein that can be translocated across the membranes of subsequently added rat liver mitochondria. Translocation is accompanied by proteolytic processing to convert the 34.5-kilodalton precursor to the 32-kilodalton mature form of the subunit. The N-terminal sequence of the mature alpha-subunit from the GTP-specific isozyme has been determined by sequential Edman degradation and compared with the amino acid sequence deduced from the cDNA. This confirms that the cloned sequence encodes the GTP-specific alpha-subunit, and establishes that the point of cleavage is between histidyl and glycyl residues and that the signal sequence consists of 27 residues. The signal sequence shares characteristics of other mitochondrial targeting sequences that have been elucidated (largely of yeast mitochondrial precursors), including the potential to form an amphiphilic helix. Import is dependent upon the presence of ATP and is inhibited by compounds that diminish mitochondrial membrane potential. Translocation of the precursor is effective for precursor produced by the reticulocyte translation system, but is not seen for the product that is translated by a wheat germ extract, indicating that the latter may lack a factor or component that is necessary for the targeting and import process.