Yano M, Kanazawa M, Terada K, Takeya M, Hoogenraad N, Mori M
Department of Molecular Genetics, Kumamoto University School of Medicine, Kumamoto 862, Japan.
J Biol Chem. 1998 Oct 9;273(41):26844-51. doi: 10.1074/jbc.273.41.26844.
The mitochondrial import receptor translocase of the outer membrane of mitochondria (Tom20) consists of five segments, an N-terminal membrane-anchor segment, a linker segment rich in charged amino acids, a tetratricopeptide repeat motif, a glutamine-rich segment, and a C-terminal segment. To assess the role of each segment, four C-terminally truncated mutants of the human receptor (hTom20) were constructed, and the effect of their overexpression in COS-7 cells was analyzed. Expression of a mutant lacking the tetratricopeptide repeat motif inhibited preornithine transcarbamylase (pOTC) import to the same extent as the wild-type receptor. Thus, overexpression of the membrane-anchor and the linker segments is sufficient for the inhibition of import. Expression of either the wild-type receptor or a mutant lacking the C-terminal end of 20 amino acid residues stimulated import of pOTC-green fluorescent protein (GFP), a fusion protein in which the presequene of pOTC was fused to green fluorescent protein. On the other hand, expression of mutants lacking either the glutamine-rich segment or larger deletions inhibited pOTC-GFP import. In vitro import of pOTC was inhibited by the wild-type hTom20 and the mutant lacking the C-terminal end, but much less strongly by the mutant lacking the glutamine-rich segment. On the other hand, import of pOTC-GFP was little affected by any of the forms of hTom20. In binding assays, pOTC binding to hTom20 was only moderately decreased by the deletion of the glutamine-rich segment, whereas pOTC-GFP binding was completely lost by this deletion. Binding of pOTCN-GFP a construct that contains an additional 58 N-terminal residues of mature OTC, resembled that of pOTC. All of these results indicate that the region 106-125 containing the glutamine-rich segment of hTom20 is essential for binding and import stimulation in vivo of pOTC-GFP and for inhibition of in vitro import of pOTC. The results also indicate that this region is important for mitochondrial aggregation. The different behaviors of pOTC and the pOTC-GFP chimera toward hTom20 mutants is explicable on the basis of the conformation of the precursor proteins.
线粒体外膜转位酶的线粒体导入受体（Tom20）由五个部分组成，一个N端膜锚定部分、一个富含带电荷氨基酸的连接部分、一个四肽重复基序、一个富含谷氨酰胺的部分和一个C端部分。为了评估每个部分的作用，构建了人类受体（hTom20）的四个C端截短突变体，并分析了它们在COS-7细胞中过表达的影响。缺乏四肽重复基序的突变体的表达对鸟氨酸氨甲酰基转移酶前体（pOTC）导入的抑制程度与野生型受体相同。因此，膜锚定部分和连接部分的过表达足以抑制导入。野生型受体或缺乏20个氨基酸残基C端的突变体的表达刺激了pOTC-绿色荧光蛋白（GFP）的导入，pOTC-绿色荧光蛋白是一种融合蛋白，其中pOTC的前序列与绿色荧光蛋白融合。另一方面，缺乏富含谷氨酰胺部分或更大缺失的突变体的表达抑制了pOTC-GFP的导入。pOTC的体外导入受到野生型hTom20和缺乏C端的突变体的抑制，但受到缺乏富含谷氨酰胺部分的突变体的抑制程度要小得多。另一方面，pOTC-GFP的导入几乎不受任何形式的hTom20的影响。在结合试验中，富含谷氨酰胺部分的缺失仅适度降低了pOTC与hTom20的结合，而这种缺失完全丧失了pOTC-GFP的结合。pOTCN-GFP（一种包含成熟OTC额外58个N端残基的构建体）的结合与pOTC的结合相似。所有这些结果表明，hTom20富含谷氨酰胺部分的106-125区域对于pOTC-GFP在体内的结合和导入刺激以及pOTC体外导入的抑制至关重要。结果还表明该区域对于线粒体聚集很重要。基于前体蛋白的构象，可以解释pOTC和pOTC-GFP嵌合体对hTom20突变体的不同行为。
