In vivo kinetics of protein targeting to the endoplasmic reticulum determined by site-specific phosphorylation
In vivo kinetics of protein targeting to the endoplasmic reticulum determined by site-specific phosphorylation
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DOI：
10.1093/emboj/19.24.6704
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发表时间：
2000-12-15
期刊：
影响因子：
11.4
通讯作者：
Spiess, M
中科院分区：
文献类型：
--
作者：
Goder, V;Crottet, P;Spiess, M
我们开发了一种新的检测方法，通过插入蛋白激酶a磷酸化的短一致序列来检测蛋白质结构域的细胞质定位。在转染的COS-1细胞中，该序列仅在暴露于细胞质溶胶时才被[P-32]磷酸盐有效地标记，而在转运到内质网管腔时则不会。因此，该序列的磷酸化状态可用于确定膜蛋白的拓扑结构。这种检测方法具有足够的灵敏度，甚至可以检测到具有反向信号锚定序列的膜蛋白的n端结构域的短暂胞质暴露。每个新合成的多肽的磷酸化程度反映了暴露于细胞质的时间，这取决于n端的翻译，靶向和易位。通过改变n端结构域的长度或操纵翻译速率，确定了蛋白质靶向是快速的，只需要几秒钟。据估计，n端易位率约为翻译率的1.6倍。
We have developed a novel assay to detect the cytosolic localization of protein domains by inserting a short consensus sequence for phosphorylation by protein kinase A. In transfected COS-1 cells, this sequence was labeled efficiently with [P-32]phosphate only when exposed to the cytosol and not when translocated into the lumen of the endoplasmic reticulum. The phosphorylation state of this sequence can therefore be used to determine the topology of membrane proteins. This assay is sufficiently sensitive to detect even the transient cytosolic exposure of the N-terminal domain of a membrane protein with a reverse signal-anchor sequence. The extent of phosphorylation per newly synthesized polypeptide was shown to reflect the time of exposure to the cytosol, which depends on translation, targeting and translocation of the N-terminus. By altering the length of the N-terminal domain or manipulating the translation rate, it was determined that protein targeting is rapid and requires only a few seconds. The rate of N-terminal translocation was estimated to be similar to1.6 times the rate of translation.