Oatey P B, Lumb M J, Danpure C J
MRC Laboratory for Molecular Cell Biology, University College London, UK.
Eur J Biochem. 1996 Oct 15;241(2):374-85. doi: 10.1111/j.1432-1033.1996.00374.x.
The molecular basis of the variable species-specific peroxisomal and/or mitochondrial targeting of the enzyme alanine-glyoxylate aminotransferase 1 (AGT) has been studied in human fibroblasts by confocal immunofluorescence microscopy after intranuclear microinjection of various human, rabbit, marmoset, and feline AGT cDNA constructs. The expression of full-length human and rabbit AGT cDNA led to an exclusively peroxisomal distribution of AGT. However, the distribution of feline and marmoset AGT depended on the cDNA construct injected. In both species, injection of the short cDNAs (from transcripts that occur naturally in marmoset liver but not in feline liver) led to an exclusively peroxisomal distribution. However, injection of the long cDNAs (from transcripts that occur naturally in both species) led to most of the AGT being targeted to the mitochondria and only a small, yet significant, fraction to the peroxisomes. Reintroduction of the 'ancestral' first potential translation initiation site into human AGT cDNA led to an 'ancestral' distribution of AGT (i.e. both mitochondrial and peroxisomal). Deletion of the second potential translation start site from the long feline cDNA led to a distribution that was almost entirely mitochondrial, which suggests that most peroxisomal AGT encoded by the long cDNA results from internal translation initiation from this site with the consequent loss of the N-terminal mitochondrial targeting sequence. Expression of rabbit cDNA and the short marmoset and feline cDNAs in cells selectively deficient in the import of peroxisomal matrix proteins showed that peroxisomal AGT in all these species is imported via the peroxisomal targeting sequence type 1 (PTS1) import pathway. The almost complete functional dominance of the N-terminal mitochondrial targeting sequence over the C-terminal PTS. which was not due to any direct interference of the former with peroxisomal import, was maintained even when the unusual PTS1 of AGT (KKL in human) was replaced by the prototypical PTS1 SKL. The results demonstrate that the major determinant of alanine-glyoxylate aminotransferase subcellular distribution in mammals is the presence or absence of the mitochondrial targeting sequence rather than the peroxisomal targeting sequence. Various strategies have arisen during the evolution of mammals to enable the exclusion of the mitochondrial targeting sequence from the newly synthesised polypeptide, all of which involve the use of alternative transcription and/or translation initiation sites.
通过对人成纤维细胞进行核内显微注射各种人、兔、狨猴和猫的丙氨酸 - 乙醛酸氨基转移酶1（AGT）cDNA构建体后，利用共聚焦免疫荧光显微镜研究了该酶可变物种特异性的过氧化物酶体和/或线粒体靶向的分子基础。全长人和兔AGT cDNA的表达导致AGT仅分布于过氧化物酶体。然而，猫和狨猴AGT的分布取决于注射的cDNA构建体。在这两个物种中，注射短cDNA（来自狨猴肝脏中天然存在但猫肝脏中不存在的转录本）导致其仅分布于过氧化物酶体。但是，注射长cDNA（来自两个物种中天然存在的转录本）导致大部分AGT靶向线粒体，只有一小部分但仍很显著的部分靶向过氧化物酶体。将“祖先”的第一个潜在翻译起始位点重新引入人AGT cDNA导致AGT出现“祖先”分布（即线粒体和过氧化物酶体都有）。从长猫cDNA中删除第二个潜在翻译起始位点导致分布几乎完全在线粒体中，这表明由长cDNA编码的大多数过氧化物酶体AGT是由该位点的内部翻译起始产生的，从而导致N端线粒体靶向序列缺失。在选择性缺乏过氧化物酶体基质蛋白导入功能的细胞中表达兔cDNA以及短的狨猴和猫cDNA表明，所有这些物种中的过氧化物酶体AGT都是通过过氧化物酶体靶向序列1（PTS1）导入途径进行导入的。即使将AGT不寻常的PTS1（人源中的KKL）替换为典型的PTS1 SKL，N端线粒体靶向序列对C端PTS的几乎完全功能优势仍然保持，这并非由于前者对过氧化物酶体导入有任何直接干扰。结果表明，哺乳动物中丙氨酸 - 乙醛酸氨基转移酶亚细胞分布的主要决定因素是线粒体靶向序列的有无，而非过氧化物酶体靶向序列。在哺乳动物进化过程中出现了各种策略，以使新合成的多肽中排除线粒体靶向序列，所有这些策略都涉及使用替代转录和/或翻译起始位点。
