F-box protein Grr1 interacts with phosphorylated targets via the cationic surface of its leucine-rich repeat
F-box protein Grr1 interacts with phosphorylated targets via the cationic surface of its leucine-rich repeat
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DOI：
10.1128/mcb.21.7.2506-2520.2001
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发表时间：
2001-04-01
影响因子：
5.3
通讯作者：
Wittenberg, C
中科院分区：
文献类型：
--
作者：
Hsiung, YG;Chang, HC;Wittenberg, C
泛素依赖的蛋白水解酶的灵活性和特异性部分是由E3泛素连接酶介导的。一类E3酶，Skp1/cullin/F-box蛋白(SCF)，其特异性来源于F-box蛋白，这是一个异质的靶蛋白识别接头家族。Grr1是SCFGrr1的F-box成分，介导与G(1)细胞周期蛋白Cln1和Cln2的磷酸化形式的相互作用。我们发现SCFGrr1与Cln2的结合依赖于其富含亮氨酸重复序列(LRR)结构域和其羧基末端。我们的Grr1 LRR结构模型预测了在特征马蹄形结构的凹面上有高密度的正电荷。我们假设，预测的凹面上的特定碱性残基对于识别磷酸化的Cln2是重要的。我们发现，将凹表面的碱性残基而不是凸表面的碱性残基转化为中性或酸性残基的点突变干扰了Grr1与Cln2的结合能力。同样的突变导致了Cln2和Gic2的稳定，也导致了GRR1失活的一系列表型，包括超极化和促进假菌丝生长。令人惊讶的是，相同的残基对Grr1在HXT1或AGP1的营养调控转录中的作用并不重要。我们得出结论，Grr1 LRR凹面的阳离子性质对于识别SCFGrr1的磷酸化靶标至关重要，但Grr1的其他功能需要Grr1的其他性质。
The flexibility and specificity of ubiquitin-dependent proteolysis are mediated, in part, by the E3 ubiquitin ligases. One class of E3 enzymes, SKp1/cullin/F-box protein (SCF), derives its specificity from F-box proteins, a heterogeneous family of adapters for target protein recognition. Grr1, the F-box component of SCFGrr1 mediates the interaction with phosphorylated forms of the G(1) cyclins Cln1 and Cln2. We show that binding of Cln2 by SCFGrr1 was dependent upon its leucine-rich repeat (LRR) domain and its carboxy terminus. Our structural model for the Grr1 LRR predicted a high density of positive charge on the concave surface of the characteristic horseshoe structure. We hypothesized that specific basic residues on the predicted concave surface are important for recognition of phosphorylated Cln2. We show that point mutations that converted the basic residues on the concave surface but not those on the convex surface to neutral or acidic residues interfered with the capacity of Grr1 to bind to Cln2. The same mutations resulted in the stabilization of Cln2 and Gic2 and also in a spectrum of phenotypes characteristic of inactivation of GRR1, including hyperpolarization and enhancement of pseudohyphal growth. It was surprising that the same residues were not important for the role of Grr1 in nutrient-regulated transcription of HXT1 or AGP1, We concluded that the cationic nature of the concave surface of the Grr1 LRR is critical for the recognition of phosphorylated targets of SCFGrr1 but that other properties of Grr1 are required for its other functions.