Structure and function of multifunctional protein complex regulating cell polarity

细胞极化是细胞在特定时间点沿确定的轴建立沿着不对称性的基本过程。极化通常通过响应于空间线索的特定蛋白质、核酸或细胞器的不对称分布而发生。在芽殖酵母酿酒酵母中，这种极化诱导不对称生长以形成芽，芽成为子细胞。BUD 32的缺失导致特异性地在二倍体细胞中的随机出芽。Bud 32 p是一种非典型激酶，参与Akt/PKB的酵母同系物Sch 9 p激酶的信号级联。在这项研究中，我们发现，Bud 32复合体参与双极出芽通过调节Bud 9 p的本地化。Bud 32 p的激酶活性对EKC/KEOPS复合物的功能至关重要，也是双极芽位点选择所必需的。然而，Bud 32 p在磷酸化位点的突变Sch 9 p并不影响双极出芽。BUD 9是EKC/KEOPS各缺失突变体随机出芽所必需的。Bud 9 p的不对称定位依赖于复合物，而Bud 8 p和Rax 2 p则不依赖于复合物。RAX 2基因位于EKC/KEOPS基因的上游，调控双极出芽。Split-GFP分析显示，Bud 9 p与Rax 2 p在出芽母细胞中的出生疤痕处发生物理相互作用。这些观察结果表明，Rax 2 p与Bud 8 p和Bud 9 p的相互作用可能有助于将双极标志移位到正确的位点。我们的结论是，EKC/KEOPS复合物是专门参与调节Bud 9 p定位下游的Rax 1 p/Rax 2 p。

Cell polarization is a fundamental process by which cells establish asymmetry along a defined axis at a specific time point. The polarization generally occurs by an asymmetric distribution of specific proteins, nucleic acid or organelles in response to spatial cues. In the budding yeast Saccharomyces cerevisiae, such polarization induces asymmetric growth to form a bud which becomes the daughter cell. The deletion of BUD32 causes a random budding specifically in diploid cells. Bud32p, an atypical kinase, is involved in a signaling cascade of Sch9p kinase, a yeast homolog of Akt/PKB. In this study, we revealed that the Bud32 complex is involved in bipolar budding by regulating the localization of Bud9p. The kinase activity of Bud32p, which is essential for the functions of the EKC/KEOPS complex, was also required for bipolar bud site selection. However, the mutation of Bud32p at the phosphorylation site by Sch9p did not affect bipolar budding. BUD9 was necessary for random budding in the each deletion mutant of EKC/KEOPS components. The asymmetric localization of Bud9p was dependent on the complex, but Bud8p and Rax2p were not. RAX2 was genetically upstream of EKC/KEOPS genes for the regulation of bipolar budding. Split-GFP analysis revealed that Bud9p physically interacted with Rax2p at the birth scar in budded mother cells. These observations suggest that the interaction of Rax2p with Bud8p and Bud9p may contribute to the translocation of bipolar landmarks to the correct sites. We concluded that the EKC/KEOPS complex is specifically involved in the regulation of Bud9p localization downstream of Rax1p/Rax2p.