In vitro transport of membrane proteins to peroxisomes by shuttling receptor Pex19p
In vitro transport of membrane proteins to peroxisomes by shuttling receptor Pex19p
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DOI：
10.1074/jbc.m509819200
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发表时间：
2006-01-06
影响因子：
4.8
通讯作者：
Fujiki, Y
中科院分区：
文献类型：
--
作者：
Matsuzono, Y;Fujiki, Y
包含299个氨基酸的过氧化物酶Pex 19 p在过氧化物酶体膜组装中起作用。我们在这里开发了一个无细胞系统，用于将膜蛋白转运到过氧化物酶体。Pex 19 p与多种膜过氧化物酶相互作用，包括参与基质蛋白输入的其他膜生物生成过氧化物酶Pex 16 p和Pex 26 p。将无细胞合成的S-35标记的Pex 19 p以ATP依赖性方式靶向含有来自中国仓鼠卵巢- K1细胞的过氧化物酶体以及从大鼠肝脏分离的过氧化物酶体的亚细胞级分。当在含有分离的过氧化物酶体的反应混合物中与S-35-Pex 19 p一起孵育时，用体外合成的具有两个跨膜片段和C-尾锚型S-35-Pex 26 p的S-35-Pex 16 p也能复制这种易位.通过碳酸钠提取法评估，将转运的S-35-Pex 16 p和S-35-Pex 26 p整合到膜中。在不存在ATP的情况下孵育后，过氧化物酶体相关的和部分Na 2CO 3抗性的S-35-Pex 19 p被释放到胞质组分中，而S-35-Pex 16 p和S-35-Pex 26 p保留在膜中。此外，不仅35 S-Pex 19 p而且35 S-Pex 19 p与S-35-Pex 16 p和S-35-Pex 26 p的复合物在体外与S-35-Pex 3 p结合。总之，这些结果强烈地表明，Pex 19 p以ATP和Pex 3 p依赖性方式将膜过氧化物酶从胞质溶胶易位到过氧化物酶体，然后穿梭回到胞质溶胶。
The peroxin Pex19p comprising 299 amino acids functions in peroxisomal membrane assembly. We here developed a cell- free system for transport of membrane proteins to peroxisomes. Pex19p interacts with multiple membrane peroxins, including other membrane biogenesis peroxins, Pex16p and Pex26p, involved in matrix protein import. Cell- free synthesized, S-35- labeled Pex19p was targeted to subcellular fractions containing peroxisomes from Chinese hamster ovary- K1 cells as well as peroxisomes isolated from rat liver in an ATP- dependent manner. Such translocation was also reproduced with in vitro synthesized S-35- Pex16p with two transmembrane segments and C- tail anchor- type S-35- Pex26p, upon incubation with S-35- Pex19p in the reaction mixtures containing isolated peroxisomes. The transported S-35- Pex16p and S-35- Pex26p were integrated into membranes as assessed by the sodium carbonate extraction method. Peroxisome- associated and partly Na2CO3- resistant S-35- Pex19p was released to the cytosolic fraction upon incubation in the absence of ATP, whereas S-35- Pex16p and S-35- Pex26p remained in the membranes. Furthermore, not only 35S- Pex19p but also 35S- Pex19p complexes each with S-35- Pex16p and S-35- Pex26p were bound to S-35- Pex3p in vitro. Together, these results strongly suggested that Pex19p translocates the membrane peroxins from the cytosol to peroxisomes in an ATP- and Pex3p- dependent manner and then shuttles back to the cytosol.