Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel.
Biochem Biophys Res Commun. 2010 Apr 9;394(3):553-7. doi: 10.1016/j.bbrc.2010.03.017. Epub 2010 Mar 6.
The Arf1 GTPase-activating protein ArfGAP1 regulates vesicular traffic through the COPI system. This protein consists of N-terminal catalytic domain, lipid packing sensors (the ALPS motifs) in the central region, and a carboxy part of unknown function. The carboxy part contains several diaromatic sequences that are reminiscent of motifs known to interact with clathrin adaptors. In pull-down experiments using GST-fused peptides from rat ArfGAP1, a peptide containing a (329)WETF sequence interacted strongly with clathrin adaptors AP1 and AP2, whereas a major coatomer-binding determinant was identified within the extreme carboxy terminal peptide ((405)AADEGWDNQNW). Mutagenesis and peptide competition experiments revealed that this determinant is required for coatomer binding to full-length ArfGAP1, and that interaction is mediated through the delta-subunit of the coatomer adaptor-like subcomplex. Evidence for a role of the carboxy motif in ArfGAP1-coatomer interaction in vivo is provided by means of a reporter fusion assay. Our findings point to mechanistic differences between ArfGAP1 and the other ArfGAPs known to function in the COPI system.
Arf1 GTPase 激活蛋白 ArfGAP1 通过 COPI 系统调节囊泡运输。该蛋白由 N 端催化结构域、中央区域的脂质包装传感器（ALPS 基序）和未知功能的羧基部分组成。羧基部分包含几个二芳基序列，这些序列让人联想到已知与网格蛋白衔接蛋白相互作用的基序。在使用 GST 融合肽的大鼠 ArfGAP1 下拉实验中，含有 (329)WETF 序列的肽与网格蛋白衔接蛋白 AP1 和 AP2 强烈相互作用，而主要的衣被蛋白结合决定簇则位于极端羧基末端肽内 ((405)AADEGWDNQNW)。突变和肽竞争实验表明，该决定簇是衣被蛋白与全长 ArfGAP1 结合所必需的，并且通过衣被蛋白衔接子样亚基复合物的 δ-亚基介导相互作用。通过报告基因融合测定法提供了羧基基序在体内 ArfGAP1-衣被蛋白相互作用中的作用的证据。我们的发现指出了 ArfGAP1 和已知在 COPI 系统中发挥作用的其他 ArfGAP 之间的机制差异。
