A GFP-based assay reveals a role for RHD3 in transport between the endoplasmic reticulum and Golgi apparatus
A GFP-based assay reveals a role for RHD3 in transport between the endoplasmic reticulum and Golgi apparatus
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DOI：
10.1046/j.1365-313x.2003.01969.x
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发表时间：
2004-02-01
期刊：
影响因子：
7.2
通讯作者：
Moore, I
中科院分区：
文献类型：
--
作者：
Zheng, HQ;Kunst, L;Moore, I
我们描述了一种分泌型的水杉绿色荧光蛋白(SecGFP)在拟南芥细胞膜运输的非侵入性活细胞检测中的应用。我们发现，与聚集在内质网(ER)中的GFP-HDEL相比，secGFP在运输到质外体时会产生微弱的荧光信号。细胞质外体中secGFP的荧光强度随着幼苗在pH为8.1的缓冲液中的生长而增强，表明质外体的pH至少是表达secGFP的幼苗荧光强度较低的部分原因。无论是通过BFA处理还是通过基因干预抑制SecGFP在ER和质膜(PM)之间的转运，都会导致secGFP在细胞内积累增加，同时secGFP的荧光强度增加。因此，secGFP为拟南芥的膜运输和内膜组织的正向和反向遗传分析提供了一个有价值的工具。利用这种定量亚致死性干扰SecGFP运输的方法，我们确定了根毛缺陷3(RHD3)在内质网和高尔基体之间分泌和高尔基标记运输中的作用。
We describe the use of a secreted form of Aequoria victoria green fluorescent protein (secGFP) in a non-invasive live cell assay of membrane traffic in Arabidopsis thaliana. We show that in comparison to GFP-HDEL, which accumulates in the endoplasmic reticulum (ER), secGFP generates a weak fluorescence signal when transported to the apoplast. The fluorescence of secGFP in the apoplast can be increased by growth of seedlings on culture medium buffered at pH 8.1, suggesting that apoplastic pH is responsible, at least in part, for the low fluorescence intensity of seedlings expressing secGFP. Inhibition of secGFP transport between the ER and plasma membrane (PM), either by Brefeldin A (BFA) treatment or by genetic intervention results in increased intracellular secGFP accumulation accompanied by an increase in the secGFP fluorescence intensity. secGFP thus provides a valuable tool for forward and reverse genetic analysis of membrane traffic and endomembrane organisation in Arabidopsis. Using this assay for quantitative sublethal perturbation of secGFP transport, we identify a role for root hair defective 3 (RHD3) in transport of secreted and Golgi markers between the ER and the Golgi apparatus.