Distinct and overlapping roles for AP-1 and GGAs revealed by the "knocksideways" system.
Distinct and overlapping roles for AP-1 and GGAs revealed by the "knocksideways" system.
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DOI：
10.1016/j.cub.2012.07.012
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发表时间：
2012-09-25
期刊：
影响因子：
9.2
通讯作者：
Robinson, Margaret S.
中科院分区：
文献类型：
--
作者：
Hirst, Jennifer;Borner, Georg H. H.;Antrobus, Robin;Peden, Andrew A.;Hodson, Nicola A.;Sahlender, Daniela A.;Robinson, Margaret S.
虽然衔接蛋白复合物1（AP-1）和高尔基体定位的含γ耳的ADP核糖基化因子结合蛋白（GGA）都是网格蛋白介导的细胞内运输的衔接蛋白，但它们介导的途径以及它们之间的关系仍然是悬而未决的问题。为了梳理AP-1和GGA的功能，我们使用“敲侧”系统快速灭活每个接头，然后比较来自对照和敲侧细胞的网格蛋白包被囊泡（CCV）组分的蛋白质组成。AP-1的侧撞导致了CCV中GGA 2的戏剧性和意想不到的损失。超过30种其他外周膜蛋白和超过30种跨膜蛋白也被耗尽，包括遗传性疾病中的几种突变，表明AP-1作为细胞内CCV形成的关键。相比之下，GGA 2敲除只影响溶酶体水解酶及其受体。我们认为，至少有两个群体的细胞内CCV：一个包含GGA和AP-1的顺行运输和另一个包含AP-1的逆行运输。我们的研究表明，knocksideways和蛋白质组学是研究蛋白质功能的强大组合，可以潜在地用于许多不同类型的蛋白质。GGA 2敲除主要消耗水解酶及其受体，GGA 2依赖于AP-1掺入CCV。AP-1作为细胞内CCV形成的关键，是双向的
Although adaptor protein complex 1 (AP-1) and Golgi-localized, γ ear-containing, ADP-ribosylation factor-binding proteins (GGAs) are both adaptors for clathrin-mediated intracellular trafficking, the pathways they mediate and their relationship to each other remain open questions. To tease apart the functions of AP-1 and GGAs, we rapidly inactivated each adaptor using the “knocksideways” system and then compared the protein composition of clathrin-coated vesicle (CCV) fractions from control and knocksideways cells. The AP-1 knocksideways resulted in a dramatic and unexpected loss of GGA2 from CCVs. Over 30 other peripheral membrane proteins and over 30 transmembrane proteins were also depleted, including several mutated in genetic disorders, indicating that AP-1 acts as a linchpin for intracellular CCV formation. In contrast, the GGA2 knocksideways affected only lysosomal hydrolases and their receptors. We propose that there are at least two populations of intracellular CCVs: one containing both GGAs and AP-1 for anterograde trafficking and another containing AP-1 for retrograde trafficking. Our study shows that knocksideways and proteomics are a powerful combination for investigating protein function, which can potentially be used on many different types of proteins. ► AP-1 knocksideways depletes ∼100 proteins from clathrin-coated vesicles (CCVs) ► GGA2 knocksideways mainly depletes hydrolases and their receptors ► GGA2 depends on AP-1 for incorporation into CCVs ► AP-1 acts as a linchpin for intracellular CCV formation and is bidirectional
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影响因子：
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