Detection of transient in vivo interactions between substrate and transporter during protein translocation into the endoplasmic reticulum
Detection of transient in vivo interactions between substrate and transporter during protein translocation into the endoplasmic reticulum
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DOI：
10.1091/mbc.10.2.329
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发表时间：
1999-02-01
影响因子：
3.3
通讯作者：
Johnsson, N
中科院分区：
文献类型：
--
作者：
D端nnwald, M;Varshavsky, A;Johnsson, N
分裂泛素技术用于检测活细胞中的瞬时蛋白质相互作用。将泛素（ubiquitin，Ub）的N端半部分N-ub与酿酒酵母（Saccharomycescerevisiae）内质网中蛋白质转运机制的组成部分Sec 62 p融合。C-ub是Ub的C-末端的一半，与信号序列的C-末端融合。从Ub的两半重建准天然Ub结构，以及在C-ub的C末端由Ub特异性蛋白酶产生的切割，用作与N-ub和C-ub连接的两种测试蛋白之间的接近度的量度。使用该测定，我们表明，Sec 62 p是空间接近的信号序列的前原α因子在体内。这种接近仅限于紧跟着信号序列的新生多肽链。此外，接近程度取决于信号序列的性质。携带转化酶信号序列的C-ub融合物导致用N-ub-Sec 62 p进行的Ub重建比携带前原-α-因子信号序列的其他相同测试蛋白低得多。在该测定中，Sec 62 p的无活性衍生物未能与信号序列相互作用。这些体内发现与Sec 62 p是信号序列结合复合物的一部分一致。
The split-ubiquitin technique was used to detect transient protein interactions in living cells. N-ub, the N-terminal half of ubiquitin (Ub), was fused to Sec62p, a component of the protein translocation machinery in the endoplasmic reticulum of Saccharomyces cerevisiae. C-ub, the C-terminal half of Ub, was fused to the C terminus of a signal sequence. The reconstitution of a quasi-native Ub structure from the two halves of Ub, and the resulting cleavage by Ub-specific proteases at the C terminus of C-ub, serve as a gauge of proximity between the two test proteins linked to N-ub and C-ub. Using this assay, we show that Sec62p is spatially close to the signal sequence of the prepro-alpha-factor in vivo. This proximity is confined to the nascent polypeptide chain immediately following the signal sequence. In addition, the extent of proximity depends on the nature of the signal sequence. C-ub fusions that bore the signal sequence of invertase resulted in a much lower Ub reconstitution with N-ub-Sec62p than otherwise identical test proteins bearing the signal sequence of prepro-alpha-factor. An inactive derivative of Sec62p failed to interact with signal sequences in this assay. These in vivo findings are consistent with Sec62p being part of a signal sequence-binding complex.