The γ/σ1 and α/σ2 hemicomplexes of clathrin adaptors AP-1 and AP-2 harbor the dileucine recognition site
The γ/σ1 and α/σ2 hemicomplexes of clathrin adaptors AP-1 and AP-2 harbor the dileucine recognition site
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DOI：
10.1091/mbc.e07-01-0012
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发表时间：
2007-05-01
影响因子：
3.3
通讯作者：
Kornfeld, Stuart
中科院分区：
文献类型：
--
作者：
Doray, Balraj;Lee, Intaek;Kornfeld, Stuart
网格蛋白接头 AP-1 和 AP-2 通过两种类型的基序结合货物蛋白：基于酪氨酸的 Yxx phi 和基于双亮氨酸的 [DE]XXXL[LI]。尽管Yxx phi 基序与AP-1 或AP-2 的mu 亚基结合已得到充分证实，但据报道，双亮氨酸基序与这些衔接子的mu 或β 亚基以及AP-1 的gamma/sigma 1 半复合物结合。为了澄清这一争议，AP-1 和 AP-2 的各个亚基在昆虫细胞中单独表达并以半复合物形式表达，并将它们用于谷胱甘肽 S-转移酶 Pull-down 测定以确定它们的结合特性。我们报告说，gamma/sigma 1 或 alpha/sigma 2 半复合物与几种蛋白质的双亮氨酸基序的结合相当牢固，而 beta 1/mu 1 和 beta 2/mu 2 半复合物以及单个 β 或 mu 亚基的结合非常弱或无法检测到。伽马/西格玛 1 和 α/西格玛 2 半复合物在对特定双亮氨酸基序的偏好方面表现出显着差异。最引人注目的是，-4 位的天冬氨酸会损害与 γ/σ 1 半复合物的结合，而对与 α/σ 2 的结合影响最小。与 α/σ 2 半复合物的结合与基于双亮氨酸的分选信号介导的体内内化之间存在极好的相关性。这些发现为 D/EXXXXL[LI] 介导的分选信号的运输机制提供了新的见解。
The clathrin adaptors AP-1 and AP-2 bind cargo proteins via two types of motifs: tyrosine-based Yxx phi and dileucine-based [DE]XXXL[LI]. Although it is well established that Yxx phi motifs bind to the mu subunits of AP-1 or AP-2, dileucine motifs have been reported to bind to either the mu or beta subunits of these adaptors as well as the gamma/sigma 1 hemicomplex of AP-1. To clarify this controversy, the various subunits of AP-1 and AP-2 were expressed individually and in hemicomplex form in insect cells, and they were used in glutathione S-transferase pull-down assays to determine their binding properties. We report that the gamma/sigma 1 or alpha/sigma 2 hemicomplexes bound the dileucine-based motifs of several proteins quite strongly, whereas binding by the beta 1/mu 1 and beta 2/mu 2 hemicomplexes, and the individual beta or mu subunits, was extremely weak or undetectable. The gamma/sigma 1 and alpha/sigma 2 hemicomplexes displayed substantial differences in their preference for particular dileucine-based motifs. Most strikingly, an aspartate at position -4 compromised binding to the gamma/sigma 1 hemicomplex, whereas minimally affecting binding to alpha/sigma 2. There was an excellent correlation between binding to the alpha/sigma 2 hemicomplex and in vivo internalization mediated by the dileucine-based sorting signals. These findings provide new insights into the trafficking mechanisms of D/EXXXL[LI]-mediated sorting signals.