ABSTRACT

Cytokinesis is the final stage of the cell cycle which separates cellular constituents to produce two daughter cells. Using the fission yeast Schizosaccharomyces pombe we have investigated the role of various classes of proteins involved in this process. Central to these is anillin/Mid1p which forms a ring-like structure at the cell equator that predicts the site of cell separation through septation in fission yeast. Here we demonstrate a direct physical interaction between Mid1p and the endosomal sorting complex required for transport (ESCRT)-associated protein Vps4p, a genetic interaction of the mid1 and vps4 genes essential for cell viability, and a requirement of Vps4p for the correct cellular localization of Mid1p. Furthermore, we show that Mid1p is phosphorylated by aurora kinase, a genetic interaction of the mid1 and the aurora kinase ark1 genes is essential for cell viability, and that Ark1p is also required for the correct cellular localization of Mid1p. We mapped the sites of phosphorylation of Mid1p by human aurora A and the polo kinase Plk1 and assessed their importance in fission yeast by mutational analysis. Such analysis revealed serine residues S332, S523 and S531 to be required for Mid1p function and its interaction with Vps4p, Ark1p and Plo1p. Combined these data suggest a physical interaction between Mid1p and Vps4p important for cytokinesis, and identify phosphorylation of Mid1p by aurora and polo kinases as being significant for this process.

摘要

细胞分裂是细胞周期的最后阶段，它分离细胞成分以产生两个子细胞。使用裂殖酵母粟酒裂殖酵母，我们研究了参与该过程的各类蛋白质的作用。这些的核心是 anillin/Mid1p，它在细胞赤道形成环状结构，预测通过裂殖酵母中的分隔进行细胞分离的部位。在这里，我们展示了 Mid1p 和运输所需的内体分选复合物 (ESCRT) 相关蛋白 Vps4p 之间的直接物理相互作用，mid1 和 vps4的遗传相互作用细胞活力所必需的基因，以及 Vps4p 对 Mid1p 正确细胞定位的要求。此外，我们表明 Mid1p 被极光激酶磷酸化，这是mid1和极光激酶ark1的遗传相互作用基因对于细胞活力至关重要，并且 Ark1p 也是 Mid1p 正确细胞定位所必需的。我们绘制了人类极光 A 和 polo 激酶 Plk1 对 Mid1p 磷酸化的位点，并通过突变分析评估了它们在裂殖酵母中的重要性。这种分析揭示了 Mid1p 功能及其与 Vps4p、Ark1p 和 Plo1p 的相互作用所需的丝氨酸残基 S332、S523 和 S531。结合这些数据表明，Mid1p 和 Vps4p 之间的物理相互作用对胞质分裂很重要，并确定极光和 polo 激酶对 Mid1p 的磷酸化对该过程具有重要意义。