Many protein-modifying enzymes recognize their substrates via docking motifs, but the range of functionally permissible motif sequences is often poorly defined. During eukaryotic cell division, cyclin-specific docking motifs help cyclin-dependent kinases (CDKs) phosphorylate different substrates at different stages, thus enforcing a temporally ordered series of events. In budding yeast, CDK substrates with Leu/Pro-rich (LP) docking motifs are recognized by Cln1/2 cyclins in late G1 phase, yet the key sequence features of these motifs were unknown. Here, we comprehensively analyze LP motif requirements in vivo by combining a competitive growth assay with deep mutational scanning. We quantified the effect of all single-residue replacements in five different LP motifs by using six distinct G1 cyclins from diverse fungi including medical and agricultural pathogens. The results uncover substantial tolerance for deviations from the consensus sequence, plus requirements at some positions that are contingent on the favorability of other motif residues. They also reveal the basis for variations in functional potency among wild-type motifs, and allow derivation of a quantitative matrix that predicts the strength of other candidate motif sequences. Finally, we find that variation in docking motif potency can advance or delay the time at which CDK substrate phosphorylation occurs, and thereby control the temporal ordering of cell cycle regulation. The overall results provide a general method for surveying viable docking motif sequences and quantifying their potency in vivo, and they reveal how variations in docking strength can tune the degree and timing of regulatory modifications.

许多蛋白质修饰酶通过对接基序识别其底物，但功能上允许的基序序列的范围通常定义不明确。在真核细胞分裂过程中，细胞周期蛋白特异性对接基序帮助细胞周期蛋白依赖性激酶（CDK）在不同阶段磷酸化不同的底物，从而执行一系列按时间顺序排列的事件。在出芽酵母中，具有富含 Leu/Pro (LP) 对接基序的 CDK 底物在 G1 期晚期被 Cln1/2 细胞周期蛋白识别，但这些基序的关键序列特征尚不清楚。在这里，我们通过将竞争性生长测定与深度突变扫描相结合来全面分析体内LP 基序的需求。我们通过使用来自不同真菌（包括医学和农业病原体）的六种不同的 G1 细胞周期蛋白，量化了五种不同 LP 基序中所有单残基替换的效果。结果揭示了对共有序列偏差的显着耐受性，以及某些位置的要求，这些要求取决于其他基序残基的有利性。它们还揭示了野生型基序之间功能效力变化的基础，并允许推导预测其他候选基序序列强度的定量矩阵。最后，我们发现对接基序效力的变化可以提前或延迟CDK底物磷酸化发生的时间，从而控制细胞周期调节的时间顺序。总体结果提供了一种用于调查可行的对接基序序列并量化其体内效力的通用方法，并且它们揭示了对接强度的变化如何调整调节修饰的程度和时间。