The maintenance of the endoplasmic reticulum network is regulated by p47, a cofactor of p97, through phosphorylation by cdc2 kinase
The maintenance of the endoplasmic reticulum network is regulated by p47, a cofactor of p97, through phosphorylation by cdc2 kinase
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DOI：
10.1111/j.1365-2443.2005.00837.x
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发表时间：
2005-04-01
期刊：
影响因子：
2.1
通讯作者：
Murata, M
中科院分区：
文献类型：
--
作者：
Kano, F;Kondo, H;Murata, M
内质网(ER)具有典型的复杂的多边形结构，在许多类型的细胞中具有显著的三向连接。为了研究维持ER网络的机制，我们建立了半完整的中国仓鼠卵巢(CHO)细胞(CHO细胞)的ER分解和重组试验，这些细胞结构性地表达热休克蛋白-47和绿色荧光蛋白(GFP-HSP47)作为ER标记(称为CHO-HSP细胞)。使用这些分析方法，我们发现内质网的维持需要胞浆和三磷酸腺苷/鸟苷5‘-三磷酸(ATP/GTP)的水解，而不是肌动蛋白细丝或微管。我们还发现，无论是在用高盐溶液清洗半完整细胞后加入N-乙基马来酰亚胺处理的胞浆，还是在诺康唑处理的半完整CHO-HSP细胞中加入有丝分裂胞浆，内质网都会被破坏。通过加入间期胞浆，对有丝分裂液引起的内质网损伤进行了改造。此外，我们还发现p97的辅因子p47对于维持ER网络是必不可少的，并且cdc2激酶对p47的磷酸化导致有丝分裂的胞浆破坏ER网络。综上所述，这些结果表明，ER网络的维持需要一个由p97/P47介导的膜融合过程，细胞周期依赖性的ER网络的形态变化是通过p47的磷酸化/去磷酸化来调节的。
The endoplasmic reticulum (ER) has a characteristic complex polygonal structure with hallmark three-way junctions in many types of cells. To investigate the mechanisms responsible for maintaining the ER network, we established ER disassembly and reassembly assays in semi-intact Chinese hamster ovary (CHO) cells that constitutively expressed heat shock protein-47 fused to the green fluorescent protein (GFP-HSP47) as an ER marker (the cells are referred to as CHO-HSP cells). Using these assays, we found that maintenance of the ER network required cytosol and adenosine triphosphate/guanosine 5'-triphosphate (ATP/GTP) hydrolysis, but not actin filaments or microtubules. We also showed that the ER network was disrupted upon addition of either N-ethylmaleimide-treated cytosol after washing semi-intact cells with high salt solution or mitotic cytosol in nocodazole-treated semi-intact CHO-HSP cells. The disrupted ER network induced by mitotic cytosol was reformed by the addition of interphase cytosol. In addition, we found that p47, a cofactor of p97, was essential for the maintenance of the ER network, and that phosphorylation of p47 by cdc2 kinase resulted in ER network disruption by mitotic cytosol. Taken together, these results imply that the maintenance of the ER network requires a membrane fusion process mediated by p97/p47, and that cell cycle-dependent morphological changes of the ER network are regulated through phosphorylation/dephosphorylation of p47.