Zou Jian, Friesen Helena, Larson Jennifer, Huang Dongqing, Cox Mike, Tatchell Kelly, Andrews Brenda
Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
Mol Biol Cell. 2009 Jul;20(14):3239-50. doi: 10.1091/mbc.e08-12-1255. Epub 2009 May 20.
In the budding yeast Saccharomyces cerevisiae, the G1-specific cyclin-dependent kinases (Cdks) Cln1,2-Cdc28 and Pcl1,2-Pho85 are essential for ensuring that DNA replication and cell division are properly linked to cell polarity and bud morphogenesis. However, the redundancy of Cdks and cyclins means that identification of relevant Cdk substrates remains a significant challenge. We used array-based genetic screens (synthetic genetic array or SGA analysis) to dissect redundant pathways associated with G1 cyclins and identified Bni4 as a substrate of the Pcl1- and Pcl2-Pho85 kinases. BNI4 encodes an adaptor protein that targets several proteins to the bud neck. Deletion of BNI4 results in severe growth defects in the absence of the Cdc28 cyclins Cln1 and Cln2, and overexpression of BNI4 is toxic in yeast cells lacking the Pho85 cyclins Pcl1 and Pcl2. Phosphorylation of Bni4 by Pcl-Pho85 is necessary for its localization to the bud neck, and the bud neck structure can be disrupted by overexpressing BNI4 in pcl1Deltapcl2Delta mutant cells. Our data suggest that misregulated Bni4 may bind in an uncontrolled manner to an essential component that resides at the bud neck, causing catastrophic morphogenesis defects.
在出芽酵母酿酒酵母中，G1期特异性细胞周期蛋白依赖性激酶（Cdks）Cln1、2 - Cdc28和Pcl1、2 - Pho85对于确保DNA复制和细胞分裂与细胞极性及芽形态发生正确关联至关重要。然而，Cdks和细胞周期蛋白的冗余性意味着鉴定相关的Cdk底物仍然是一项重大挑战。我们利用基于阵列的遗传筛选（合成遗传阵列或SGA分析）来剖析与G1期细胞周期蛋白相关的冗余途径，并鉴定出Bni4是Pcl1 - 和Pcl2 - Pho85激酶的底物。BNI4编码一种衔接蛋白，可将多种蛋白质靶向到芽颈。在缺乏Cdc28细胞周期蛋白Cln1和Cln2的情况下，BNI4的缺失会导致严重的生长缺陷，而在缺乏Pho85细胞周期蛋白Pcl1和Pcl2的酵母细胞中，BNI4的过表达具有毒性。Pcl - Pho85对Bni4的磷酸化是其定位于芽颈所必需的，并且在pcl1Δpcl2Δ突变细胞中过表达BNI4会破坏芽颈结构。我们的数据表明，调控失调的Bni4可能以不受控制的方式与位于芽颈的一种必需成分结合，从而导致灾难性的形态发生缺陷。
