In vivo monitoring of the recruitment and activation of AP-1 by Arf1.
In vivo monitoring of the recruitment and activation of AP-1 by Arf1.
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DOI：
10.1038/s41598-017-07493-1
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发表时间：
2017-08-02
影响因子：
4.6
通讯作者：
Lefrancois S
中科院分区：
文献类型：
--
作者：
Sauvageau E;McCormick PJ;Lefrancois S
AP-1是一种网格蛋白接头，它可以与货物蛋白胞质尾部的特定信号相互作用，将它们整合到网格蛋白包被的囊泡中进行运输。小G蛋白Arf1调节AP-1的时空募集，也驱动有利于与货物蛋白相互作用的构象变化。最近的晶体结构和体外实验强调了介导AP-1/Arf1相互作用和解锁复合物的潜在残基。我们利用生物发光共振能量转移（BRET）技术研究了Arf1/AP-1相互作用和体内AP-1构象的变化。除了晶体结构中预测的残基外，我们还发现了这种相互作用所需的新残基。我们还研究了活细胞中Arf1驱动AP-1的构象变化，发现该复合物的打开是寡聚化的先决条件。使用CRISPR/Cas9产生的Arf1敲除细胞，我们证明Arf1中的残基172是AP-1激活所必需的，并且是溶酶体蛋白prosaposin的有效分选所必需的。我们使用BRET来研究AP-1的体内活化。BRET的优点包括在其原生环境中表达已完全翻译后修饰的全长蛋白。
AP-1 is a clathrin adaptor recruited to the trans-Golgi Network where it can interact with specific signals found in the cytosolic tail of cargo proteins to incorporate them into clathrin-coated vesicles for trafficking. The small G protein Arf1 regulates the spatiotemporal recruitment of AP-1 and also drives a conformational change favoring an interaction with cargo proteins. A recent crystal structure and in vitro experiments highlighted potential residues mediating the AP-1/Arf1 interaction and the unlocking of the complex. We have used bioluminescence resonance energy transfer (BRET) to study the Arf1/AP-1 interaction and AP-1 conformational changes in vivo. We identified novel residues required for this interaction in addition to those predicted in the crystal structure. We also studied the conformational changes in AP-1 driven by Arf1 in live cells and found that opening of the complex is prerequisite for oligomerization. Using Arf1 knockout cells generated by CRISPR/Cas9, we demonstrated that residue 172 in Arf1 is necessary for AP-1 activation and is required for the efficient sorting of the lysosomal protein prosaposin. We have used BRET to study the in vivo activation of AP-1. The advantages of BRET include expressing full-length proteins in their native environment that have been fully post-translationally modified.
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影响因子：
64.5
作者：
Ren X;Farías GG;Canagarajah BJ;Bonifacino JS;Hurley JH
通讯作者：
Hurley JH
影响因子：
3.3
作者：
Meyer, DM;Crottet, P;Spiess, M
通讯作者：
Spiess, M
DOI：
10.1111/j.1600-0854.2010.01054.x
发表时间：
2010-06
期刊：
Traffic (Copenhagen, Denmark)
影响因子：
--
作者：
Jian X;Cavenagh M;Gruschus JM;Randazzo PA;Kahn RA
通讯作者：
Kahn RA
影响因子：
2.9
作者：
Antonny, B;BeraudDufour, S;Chabre, M
通讯作者：
Chabre, M
影响因子：
4
作者：
Seaman, Matthew N. J.;Harbour, Michael E.;Bright, Nicholas
通讯作者：
Bright, Nicholas