Cell-free sorting of peroxisomal membrane proteins from the endoplasmic reticulum
Cell-free sorting of peroxisomal membrane proteins from the endoplasmic reticulum
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DOI：
10.1073/pnas.1018749108
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发表时间：
2011-05-31
影响因子：
11.1
通讯作者：
Subramani, Suresh
中科院分区：
文献类型：
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作者：
Agrawal, Gaurav;Joshi, Saurabh;Subramani, Suresh
几种酵母和哺乳动物过氧化物酶体膜蛋白（PMP）通过内质网（ER）递送到过氧化物酶体。荧光显微镜显示集中组装的PMP在一个专门的领域的ER，被称为preperoxisomal ER。有人建议，preperoxisomal囊泡含有PMP芽从这个域要么融合与预先存在的过氧化物酶体或成熟成功能过氧化物酶体的摄取过氧化物酶体膜和基质蛋白。然而，这样的囊泡实体没有被确定，也不是已知的出芽过程的生化要求。我们开发了一种体外无细胞内质网出芽试验，使用巴斯德毕赤酵母，并遵循两个内源性PMPs，Pex11p和Pex3p在其内质网退出。两种PMP均共包装在漂浮在Nycodenz梯度上的ER出芽囊泡中。PMP出芽从ER依赖于ATP，温度，胞质溶胶，和Pex19p和产生preperoxisomal囊泡与不完全的补充PMP。令人惊讶的是，Pex11p出芽是独立的Pex3p，然而，出芽囊泡是缺乏的大多数的PMP，否则存在于野生型囊泡，可能代表过氧化物酶体残留物。我们的研究结果为揭示PMP从ER出芽的机制提供了一个生化平台。
Several yeast and mammalian peroxisomal membrane proteins (PMPs) are delivered to peroxisomes via the endoplasmic reticulum (ER). Fluorescence microscopy showed a focused assembly of PMPs in a specialized domain of the ER, referred to as the preperoxisomal ER. It is proposed that preperoxisomal vesicles containing PMPs bud from this domain to either fuse with preexisting peroxisomes or to mature into functional peroxisomes by uptake of peroxisomal membrane and matrix proteins. However, such vesicular entities are not identified nor are the biochemical requirements for the budding process known. We developed an in vitro cell-free ER-budding assay using Pichia pastoris and followed two endogenous PMPs, Pex11p and Pex3p during their ER exit. Both the PMPs were copackaged in the ER-budded vesicles that float on a Nycodenz gradient. PMP budding from the ER was dependent on ATP, temperature, cytosol, and Pex19p and generated preperoxisomal vesicles with an incomplete complement of PMPs. Surprisingly, Pex11p budding was independent of Pex3p; however, the budded vesicles were devoid of most of the PMPs otherwise present in the wild-type vesicles and might represent peroxisomal remnants. Our findings provide a biochemical platform to uncover the mechanism of PMP budding from the ER.