Metabolic effects of altering redundant targeting signals for yeast mitochondrial malate dehydrogenase.
Metabolic effects of altering redundant targeting signals for yeast mitochondrial malate dehydrogenase.
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DOI：
10.1006/abbi.1997.0179
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发表时间：
1997-08
影响因子：
3.9
通讯作者：
W. Small;L. McAlister-Henn
中科院分区：
文献类型：
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作者：
W. Small;L. McAlister-Henn
真核细胞含有高度同源的苹果酸脱氢酶同工酶，它们在不同的细胞内催化相同的反应。为了检验这些同工酶的代谢功能是否可以互换，我们改变了线粒体苹果酸脱氢酶(MDH1)在酵母中的细胞定位。由于先前的研究表明，从MDH1中移除靶向前序列并不能阻止线粒体在体内的输入，因此我们测试了成熟多肽氨基末端附近假定的隐蔽靶向序列的作用。该区域的三个残基被改变为其他两个酵母MDH同工酶中类似位置的残基。单独而言，这些替换不影响MDH1的活性或定位，但与前序列的缺失结合在一起，阻止了线粒体在体内的输入。以着丝粒为基础的质粒表达的MDH1胞浆形式的可测量水平较低，但与多拷贝质粒表达的正常细胞水平相当。MDH1的胞浆形式恢复了DeltaMDH1中断菌株在乙醇或醋酸盐上生长的能力，这表明MDH1的线粒体定位对其在TCA循环中的功能并不是必需的。观察到胞浆形式的MDH1的这种TCA循环功能是该同工酶所特有的，因为在deltaMDH1菌株中过表达MDH2和胞浆形式的MDH3不能恢复生长。最后，用胞浆形式的MDH1仅部分恢复了deltaMDH2中断突变体的生长，表明MDH2也可能具有独特的代谢功能。
Eukaryotic cells contain highly homologous isozymes of malate dehydrogenase which catalyze the same reaction in different cellular compartments. To examine whether the metabolic functions of these isozymes are interchangeable, we have altered the cellular localization of mitochondrial malate dehydrogenase (MDH1) in yeast. Since a previous study showed that removal of the targeting presequence from MDH1 does not prevent mitochondrial import in vivo, we tested the role of a putative cryptic targeting sequence near the amino terminus of the mature polypeptide. Three residues in this region were changed to residues present in analogous positions in the other two yeast MDH isozymes. Alone, these replacements did not affect activity or localization of MDH1 but, in combination with deletion of the presequence, prevented mitochondrial import in vivo. Measurable levels of the resulting cytosolic form of MDH1 were low with expression from a centromere-based plasmid but were comparable to normal cellular levels with expression from a multicopy plasmid. The cytosolic form of MDH1 restored the ability of a deltaMDH1 disruption strain to grow on ethanol or acetate, suggesting that mitochondrial localization of MDH1 is not essential for its function in the TCA cycle. This TCA cycle function observed for the cytosolic form of MDH1 is unique to that isozyme since overexpression of MDH2 and of a cytosolic form of MDH3 in a deltaMDH1 strain failed to restore growth. Finally, only partial restoration of growth of a deltaMDH2 disruption mutant was attained with the cytosolic form of MDH1, suggesting that MDH2 may also have unique metabolic functions.