RGS4 is arginylated and degraded by the N-end rule pathway in vitro
RGS4 is arginylated and degraded by the N-end rule pathway in vitro
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DOI：
10.1074/jbc.m001605200
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发表时间：
2000-07-28
影响因子：
4.8
通讯作者：
Varshavsky, A
中科院分区：
文献类型：
--
作者：
Davydov, IV;Varshavsky, A
N-端规则将蛋白质在体内的半衰期与其N-端残基的同一性联系起来。我们使用表达克隆筛选来寻找在网织红细胞裂解物中被泛素/蛋白酶体依赖的N末端规则通路降解的小鼠蛋白。这样确定的底物之一是RGS4，它是GTPase激活蛋白RGS家族的成员，下调特定的G蛋白。RGS4降解信号的决定因素(Degron)位于RGS4的N端，因为将半胱氨酸2转化为甘氨酸、丙氨酸或缬氨酸完全稳定了RGS4。放射化学测序表明，裂解产物RGS4的N-末端蛋氨酸被精氨酸取代。由于N-端精氨酸是一种非RGS4编码的不稳定残基，因此我们推断RGS4的降解是通过N-端蛋氨酸的去除和N-端半胱氨酸的精氨酸化而产生的。RGS家族的另一成员RGS16也被发现是N-端规则底物。在小鼠L细胞中瞬时表达的RGS4在这些细胞中存活时间较短。然而，在这种体内环境中，RGS4的靶向降解主要涉及另一种降解，因为在网织红细胞裂解物中稳定的RGS4的N端变体在L细胞中仍然不稳定。
The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. We used an expression-cloning screen to search for mouse proteins that are degraded by the ubiquitia/proteasome-dependent N-end rule pathway in a reticulocyte lysate. One substrate thus identified was RGS4, a member of the RGS family of GTPase-activating proteins that downregulate specific G proteins. A determinant of the RGS4 degradation signal (degron) was located at the N terminus of RGS4, because converting cysteine 2 to either glycine, alanine, or valine completely stabilized RGS4. Radiochemical sequencing indicated that the N-terminal methionine of the lysate-produced RGS4 was replaced with arginine. Since N-terminal arginine is a destabilizing residue not encoded by RGS4 mRNA, we conclude that the degron of RGS4 is generated through the removal of N-terminal methionine and enzymatic arginylation of the resulting N-terminal cysteine. RGS16, another member of the RGS family, was also found to be an N-end rule substrate. RGS4 that was transiently expressed in mouse L cells was short-lived in these cells. However, the targeting of RGS4 for degradation in this in vivo setting involved primarily another degron, because N-terminal variants of RGS4 that were stable in reticulocyte lysate remained unstable in L cells.