Capturing protein interactions in the secretory pathway of living cells
Capturing protein interactions in the secretory pathway of living cells
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DOI：
10.1073/pnas.0501976102
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发表时间：
2005-05-03
影响因子：
11.1
通讯作者：
Hauri, HP
中科院分区：
文献类型：
--
作者：
Nyfeler, B;Michnick, SW;Hauri, HP
分泌途径由专门负责蛋白质折叠、修饰、运输和分类的膜室组成。许多瞬时蛋白-蛋白相互作用引导转运能力蛋白通过分泌途径。在这里，我们采用基于黄色荧光蛋白（YFP）的蛋白片段互补试验（PCA）来检测活细胞分泌途径中的蛋白-蛋白相互作用。YFP片段被融合到货物受体凝集素内质网高尔基中间隔区(ERGIC -53)-53、与ERGIC-53相互作用的多凝因子缺乏蛋白MCFD2和ERGIC-53的货物糖蛋白cathepsin z上，YFP PCA分析揭示了ERGIC-53的寡聚化及其与MCFD2的相互作用。通过对ERGIC-53凝集素结构域的突变选择性地降低了YFP与cathepsin z的互补。利用YFP PCA，我们发现了ERGIC-53与cathepsin c之间碳水化合物介导的相互作用。我们得出结论，YFP PCA可以检测分泌途径中微弱和短暂的蛋白质相互作用，因此是研究蛋白质分泌过程的有力方法。该研究将PCA的应用扩展到低亲和力的碳水化合物介导的蛋白质-蛋白质相互作用。
The secretory pathway is composed of membrane compartments specialized in protein folding, modification, transport, and sorting. Numerous transient protein-protein interactions guide the transport-competent proteins through the secretory pathway. Here we have adapted the yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) to detect protein-protein interactions in the secretory pathway of living cells. Fragments of YFP were fused to the homooligomeric cargo-receptor lectin endoplasmic reticulum Golgi intermediate compartment (ERGIC)-53, to the ERGIC-53-interacting multicoagulation factor deficiency protein MCFD2, and to ERGIC-53's cargo glycoprotein cathepsin Z. YFP PCA analysis revealed the oligomerization of ERGIC-53 and its interaction with MCFD2, as well as its lectin-mediated interaction with cathepsin Z. Mutation of the lectin domain of ERGIC-53 selectively decreased YFP complementation with cathepsin Z. Using YFP PCA, we discovered a carbohydrate-mediated interaction between ERGIC-53 and cathrepsin C. We conclude that YFP PCA can detect weak and transient protein interactions in the secretory pathway and hence is a powerful approach to study luminal processes involved in protein secretion. The study extends the application of PCA to carbohydrate-mediated protein-protein interactions of low affinity.