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不对称性的基本过程。极化通常是由特定蛋白质、核酸或细胞器响应空间信号的不对称分布引起的。在出芽的酿酒酵母中，这种极化诱导不对称生长，形成芽，成为子细胞。BUD32的缺失引起二倍体细胞的随机出芽。Bud32p是一种非典型激酶，参与Akt/PKB酵母同源物Sch9p激酶的信号级联反应。在这项研究中，我们发现Bud32复合体通过调节Bud9p的定位参与了双极出芽。Bud32p的激酶活性对EKC/KEOPS复合体的功能至关重要，也是双极芽位点选择所必需的。然而，Sch9p在磷酸化位点突变Bud32p并不影响双极出芽。在EKC/KEOPS成分缺失突变体中，BUD9是随机出芽所必需的。Bud9p的不对称定位依赖于该复合体，而Bud8p和Rax2p不依赖于该复合体。RAX2是调控双极出芽的EKC/KEOPS基因的上游基因。splitgfp分析显示Bud9p在出芽母细胞的出生疤痕处与Rax2p发生物理相互作用。这些观察结果表明，Rax2p与Bud8p和Bud9p的相互作用可能有助于双极标志物向正确位置的移位。我们得出结论，EKC/KEOPS复合体特别参与调节Rax1p/Rax2p下游的Bud9p定位。

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.