Hsiung Y G, Chang H C, Pellequer J L, La Valle R, Lanker S, Wittenberg C
Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Mol Cell Biol. 2001 Apr;21(7):2506-20. doi: 10.1128/MCB.21.7.2506-2520.2001.
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 SCF(Grr1), mediates the interaction with phosphorylated forms of the G(1) cyclins Cln1 and Cln2. We show that binding of Cln2 by SCF(Grr1) 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 SCF(Grr1) but that other properties of Grr1 are required for its other functions.
泛素依赖性蛋白水解的灵活性和特异性部分由E3泛素连接酶介导。一类E3酶，即SKp1/接头蛋白/ F盒蛋白（SCF），其特异性来源于F盒蛋白，这是一类用于识别靶蛋白的异质衔接子家族。Grr1是SCF（Grr1）的F盒成分，介导与G1期细胞周期蛋白Cln1和Cln2的磷酸化形式的相互作用。我们发现SCF（Grr1）与Cln2的结合依赖于其富含亮氨酸的重复序列（LRR）结构域及其羧基末端。我们对Grr1 LRR的结构模型预测，在特征性马蹄形结构的凹面上存在高密度的正电荷。我们推测，预测凹面上的特定碱性残基对于识别磷酸化的Cln2很重要。我们发现，将凹面上而非凸面上的碱性残基突变为中性或酸性残基的点突变会干扰Grr1与Cln2结合的能力。相同的突变导致Cln2和Gic2的稳定，也导致了一系列GRR1失活特征性的表型，包括超极化和假菌丝生长增强。令人惊讶的是，相同的残基对于Grr1在营养调节的HXT1或AGP1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SCF（Grr1）的磷酸化靶标至关重要，但Grr1的其他功能还需要其其他特性。
