Substrate-binding sites of UBR1, the ubiquitin ligase of the N-end rule pathway
Substrate-binding sites of UBR1, the ubiquitin ligase of the N-end rule pathway
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DOI：
10.1074/jbc.m802583200
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发表时间：
2008-08-29
影响因子：
4.8
通讯作者：
Varshavsky, Alexander
中科院分区：
文献类型：
--
作者：
Xia, Zanxian;Webster, Ailsa;Varshavsky, Alexander
泛素依赖的蛋白分解系统称为N-末端规则途径，底物包括具有不稳定N-末端残基的蛋白质。N-识别素是该途径的泛素连接酶，包含三个底物结合位点。类型1是碱性N-末端残基(Arg、Lys和His)的专一性位点。类型2位点专用于大体积疏水性N末端残基(Trp、Phe、Tyr、Leu和Ile)。我们在这里证明了酿酒酵母唯一的N识别蛋白UBR1的1/2类型位点位于第一个位置，类似于1,950个残基的700个残基。这些位点的不同之处在于，它们可以通过基因筛查确定的突变来选择性地灭活。失活类型1位点的突变位于先前描述的类似于N-识别蛋白特有的70个残基的UBR基序中。通过荧光偏振和表面等离子体共振确定UBR1与报告多肽的1或2类不稳定的N末端残基结合，但不与稳定的N末端残基如甘氨酸结合，K-d类似于1微米M。UBR1的第三个底物结合位点针对CUP9的内部降解，CUP9是多肽输入的转录抑制因子。我们表明，先前证明的体内CUP9泛素化依赖于同源二肽与UBR1型1/2位点的结合，可以在一个完全定义的体外系统中重建。我们还发现，纯化的UBR1和CUP9是非特异性相互作用的，特异性结合(特别是涉及同源二肽与UBR1型1/2位点的结合)可以通过EF1A等伴侣或通过大分子拥挤来恢复。
Substrates of a ubiquitin-dependent proteolytic system called the N-end rule pathway include proteins with destabilizing N-terminal residues. N-recognins, the pathway's ubiquitin ligases, contain three substrate-binding sites. The type-1 site is specific for basic N-terminal residues (Arg, Lys, and His). The type-2 site is specific for bulky hydrophobic N-terminal residues (Trp, Phe, Tyr, Leu, and Ile). We show here that the type-1/2 sites of UBR1, the sole N-recognin of the yeast Saccharomyces cerevisiae, are located in the first similar to 700 residues of the 1,950-residue UBR1. These sites are distinct in that they can be selectively inactivated by mutations, identified through a genetic screen. Mutations inactivating the type-1 site are in the previously delineated similar to 70-residue UBR motif characteristic of N-recognins. Fluorescence polarization and surface plasmon resonance were used to determine that UBR1 binds, with a K-d of similar to 1 mu M, to either type-1 or type-2 destabilizing N-terminal residues of reporter peptides but does not bind to a stabilizing N-terminal residue such as Gly. A third substrate-binding site of UBR1 targets an internal degron of CUP9, a transcriptional repressor of peptide import. We show that the previously demonstrated in vivo dependence of CUP9 ubiquitylation on the binding of cognate dipeptides to the type-1/2 sites of UBR1 can be reconstituted in a completely defined in vitro system. We also found that purified UBR1 and CUP9 interact nonspecifically and that specific binding ( which involves, in particular, the binding by cognate dipeptides to the UBR1 type-1/2 sites) can be restored either by a chaperone such as EF1A or through macromolecular crowding.