Transmembrane topology of the peroxin, Pex2p, an essential component for the peroxisome assembly
Transmembrane topology of the peroxin, Pex2p, an essential component for the peroxisome assembly
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DOI：
10.1093/oxfordjournals.jbchem.a022400
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发表时间：
1999-06-01
影响因子：
2.7
通讯作者：
Fujiki, Y
中科院分区：
文献类型：
--
作者：
Harano, T;Shimizu, N;Fujiki, Y
过氧化物酶体生物合成因子，peroxin Pex 2 p，是过氧化物酶体的一种完整的膜蛋白。作为阐明Pex 2 p的结构和生物学功能的一步，我们通过在COS-7细胞中表达表位标记的大鼠Pex 2 p来确定Pex 2 p的跨膜拓扑结构。当用50 μ g/ml毛地黄皂苷透化细胞时，在C-末端用myc标记的Pex 2 p被检测为点状染色模式，在该条件下过氧化物酶体内蛋白质如PTS 1-蛋白质是外源抗体不可接近的。在相同处理后同样检测到N-末端标记的Pex 2 p。这些结果有力地表明，Pex 2 p的N-和C-末端部分都暴露于胞质溶胶中。Pex 2 p的跨膜取向也通过使用大鼠肝过氧化物酶体和Pex 2 p区域特异性抗体进行了评估。（氨基酸残基1-131）和C-末端部分（C-末端的残基226）分别特异性识别Pex 2 p和免疫沉淀完整的整个过氧化物酶体。当过氧化物酶体用蛋白酶K处理时，Pex 2 p不被抗体识别。此外，涉及双功能试剂的原位交联研究揭示了Pex 2 p的明显二聚体形式，因此，Pex 2 p通过两个跨膜片段锚定到过氧化物酶体膜，其N-和C-末端区域暴露于胞质溶胶。
The peroxisome biogenesis factor, peroxin Pex2p, is an integral membrane protein of peroxisomes. As a step toward elucidating the structure and biological function of Pex2p, we determined the transmembrane topology of Pex2p by expressing epitope-tagged rat Pex2p in COS-7 cells. Pex2p tagged with myc at the C-terminus was detected as a punctate staining pattern, when the cells were permeabilized with 50 mu g/ml of digitonin, under which conditions intraperoxisomal proteins such as PTS1-proteins are inaccessible to exogenous antibodies. N-terminally flag-tagged Pex2p was likewise detected upon the same treatment. These results strongly suggest that both the N- and C-terminal parts of Pex2p are exposed to the cytosol, The transmembrane orientation of Pex2p was also assessed by using rat liver peroxisomes and Pex2p region-specific antibodies, The two types of antibodies used, raised to the N(amino acid residues 1-131) and C-terminal part (residues 226 to the C-terminus), respectively, specifically recognized Pex2p and immunoprecipitated intact, whole peroxisomes. Pex2p was not recognized by the antibodies when the peroxisomes were treated with Proteinase K. Furthermore, in situ crosslinking studies involving bifunctional reagents revealed an apparently dimeric form of Pex2p, Therefore, Pex2p is anchored to the peroxisomal membrane by two membrane-spanning segments, with its N- and C-terminal regions exposed to the cytosol.