Identification and characterization of the human peroxin PEX3
Identification and characterization of the human peroxin PEX3
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DOI：
10.1016/s0171-9335(99)80078-8
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发表时间：
1999-06-01
影响因子：
6.6
通讯作者：
Dodt, G
中科院分区：
文献类型：
--
作者：
Soukupova, M;Sprenger, C;Dodt, G
过氧化物酶体的生物发生需要几种由PEX基因编码的过氧化物酶相互作用，并且在酵母和人之间具有良好的保守性，我们根据其与酵母不同的PEX3基因的同源性克隆了PEX3的人类cDNA，推断出的过氧化物酶体hspex3是一个过氧化物酶体膜蛋白，计算分子量为42.1 kDa。我们创建了N和c末端标记的PEX3，通过免疫荧光显微镜分析其在过氧化物酶体膜上的拓扑结构。我们的结果和一个预测的跨膜跨越区域符合假设，即hspex3是一个完整的过氧化物酶体膜蛋白，其n端在过氧化物酶体内，c端面向细胞质。法酰化过氧化物酶体膜蛋白PEX19与hspex3在人成纤维细胞中的双杂交实验中相互作用，物理相互作用可以通过两种体外转录和翻译蛋白的共免疫沉淀来证实。为了解决PEX3对过氧化物酶体膜的靶向性，研究了PEX3不同N端和c端截断融合到绿色荧光蛋白（GFP）的表达，PEX3的N端33个氨基酸是引导报告蛋白GFP到过氧化物酶体的必要和充分的，并且似乎被整合到过氧化物酶体膜中。1-16 PEX3- gfp融合蛋白的表达并没有导致过氧化物酶体的定位，但有趣的是，这个和其他几个截断的PEX3融合蛋白也定位在代表线粒体的管状和/或水泡结构上。
The biogenesis of peroxisomes requires the interaction of several peroxins, encoded by PEX genes and is well conserved between yeast and humans, We have cloned the human cDNA of PEX3 based on its homology to different yeast PEX3 genes, The deduced peroxin H sPEX3 is a peroxisomal membrane protein,vith a calculated molecular mass of 42.1 kDa. We created N- and C-terminal tagged PEX3 to assay its topology at the peroxisomal membrane by immunofluorescence microscopy. Our results and the one predicted transmembrane spanning region are in line with the assumption that H sPEX3 is an integral peroxisomal membrane protein with the N-terminus inside the peroxisome and the C-terminus facing the cytoplasm. The farnesylated peroxisomal membrane protein PEX19 interacts with H sPEX3 in a mammalian two-hybrid assay in human fibroblasts, The physical interaction could be confirmed by coimmunoprecipitation of the two in vitro transcribed and translated proteins. To address the targeting of PEX3 to the peroxisomal membrane, the expression of different N- and C-terminal PEX3 truncations fused to green fluorescent protein (GFP) was investigated in human fibroblasts, The N-terminal 33 amino acids of PEX3 were necessary and sufficient to direct the reporter protein GFP to peroxisomes and seemed to be integrated into the peroxisomal membrane. The expression of a 1-16 PEX3-GFP fusion protein did not result in a peroxisomal localization, but interestingly, this and several other truncated PEX3 fusion proteins were also localized to tubular and/or vesicular structures representing mitochondria.