Regulatory regions of the mitochondrial and cytosolic isoenzyme genes participating in the malate-aspartate shuttle.
Regulatory regions of the mitochondrial and cytosolic isoenzyme genes participating in the malate-aspartate shuttle.
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参与苹果酸-天冬氨酸穿梭的线粒体和胞质同工酶基因的调节区域。
DOI：
10.1016/s0021-9258(19)40012-4
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
K. Shimada
中科院分区：
文献类型：
--
作者：
C. Setoyama;S. Ding;B. Choudhury;T. Joh;H. Takeshima;T. Tsuzuki;K. Shimada
苹果酸-天冬氨酸穿梭由线粒体和细胞质中天冬氨酸转氨酶以及线粒体和细胞质中苹果酸脱氢酶组成，是哺乳动物将还原性物质从细胞质转运到线粒体的主要途径。为了阐明调节线粒体和细胞质间代谢协调的分子机制，我们分析了在穿梭过程中起关键作用的整套小鼠同工酶基因的5'侧调控区域。缺失分析和NIH3T3细胞体内转染实验显示，所有启动子区域都位于起始密码子上游的300个碱基对区域内。随后，使用NIH3T3细胞核提取物进行DNase I足迹分析，在这些启动子区域内鉴定了几个蛋白质结合位点。合成的低聚物含有RNA聚合酶II转录因子CTF/NFI的一致结合位点序列，竞争与细胞质天冬氨酸氨基转移酶、线粒体和细胞质苹果酸脱氢酶基因启动子区域的蛋白质结合，但不竞争与线粒体天冬氨酸氨基转移酶基因的结合。另一方面，含有Sp1一致结合位点序列的人工合成寡聚物，可以激活含有正确定位GC盒的启动子的转录，竞争与线粒体天冬氨酸转氨酶基因启动子区域结合的蛋白质。
The malate-aspartate shuttle, consisting of mitochondrial and cytosolic aspartate aminotransferase and mitochondrial and cytosolic malate dehydrogenase, is a major pathway for the transport of reducing equivalents from cytosol to mitochondria in mammals. To elucidate molecular mechanisms regulating metabolic coordination between the mitochondria and the cytosol, we analyzed the 5'-flanking regulatory regions of the complete set of mouse isoenzyme genes playing a pivotal role in the shuttle. Deletion analysis and an in vivo transfection assay, using NIH3T3 cells, revealed that all the promoter regions are located within the 300-base pair regions upstream from the initiation codon. Subsequently, DNase I footprinting analyses using NIH3T3 cell nuclear extracts led to identification of several protein binding sites within these promoter regions. A synthetic oligomer containing the consensus binding site sequence for CTF/NFI, a transcription factor for RNA polymerase II, competed for the binding of proteins to the promoter regions of cytosolic aspartate aminotransferase and mitochondrial and cytosolic malate dehydrogenase genes, but not for that of the mitochondrial aspartate amino-transferase gene. On the other hand, a synthetic oligomer containing the consensus binding site sequence for Sp1, which activates transcription from promoters containing properly positioned GC boxes, competed for protein(s) binding to the promoter region of the mitochondrial aspartate aminotransferase gene.