Verma R, Annan R S, Huddleston M J, Carr S A, Reynard G, Deshaies R J
Division of Biology, Box 156-29, California Institute of Technology, Pasadena, CA 91125, USA.
Science. 1997 Oct 17;278(5337):455-60. doi: 10.1126/science.278.5337.455.
G1 cyclin-dependent kinase (Cdk)-triggered degradation of the S-phase Cdk inhibitor Sic1p has been implicated in the transition from G1 to S phase in the cell cycle of budding yeast. A multidimensional electrospray mass spectrometry technique was used to map G1 Cdk phosphorylation sites in Sic1p both in vitro and in vivo. A Sic1p mutant lacking three Cdk phosphorylation sites did not serve as a substrate for Cdc34p-dependent ubiquitination in vitro, was stable in vivo, and blocked DNA replication. Moreover, purified phosphoSic1p was ubiquitinated in cyclin-depleted G1 extract, indicating that a primary function of G1 cyclins is to tag Sic1p for destruction. These data suggest a molecular model of how phosphorylation and proteolysis cooperate to bring about the G1/S transition in budding yeast.
G1 细胞周期蛋白依赖性激酶（Cdk）引发的 S 期 Cdk 抑制剂 Sic1p 的降解与芽殖酵母细胞周期中从 G1 期到 S 期的转变有关。一种多维电喷雾质谱技术被用于在体外和体内绘制 Sic1p 中的 G1 Cdk 磷酸化位点。一个缺少三个 Cdk 磷酸化位点的 Sic1p 突变体在体外不是 Cdc34p 依赖性泛素化的底物，在体内是稳定的，并且阻断了 DNA 复制。此外，纯化的磷酸化 Sic1p 在细胞周期蛋白缺失的 G1 提取物中被泛素化，表明 G1 细胞周期蛋白的主要功能是标记 Sic1p 以便破坏。这些数据提示了一个关于磷酸化和蛋白水解如何协同作用以实现芽殖酵母中 G1/S 转变的分子模型。
