The kinetochore, a multi-protein complex assembled on centromeres, is essential to segregate chromosomes during cell division. Deficiencies in kinetochore function can lead to chromosomal instability and aneuploidy—a hallmark of cancer cells. Kinetochore function is controlled by recruitment of regulatory proteins, many of which have been documented, however their function often remains uncharacterized and many are yet to be identified. To identify candidates of kinetochore regulation we used a proteome-wide protein association strategy in budding yeast and detected many proteins that are involved in post-translational modifications such as kinases, phosphatases and histone modifiers. We focused on the Polo-like kinase, Cdc5, and interrogated which cellular components were sensitive to constitutive Cdc5 localization. The kinetochore is particularly sensitive to constitutive Cdc5 kinase activity. Targeting Cdc5 to different kinetochore subcomplexes produced diverse phenotypes, consistent with multiple distinct functions at the kinetochore. We show that targeting Cdc5 to the inner kinetochore, the constitutive centromere-associated network (CCAN), increases the levels of centromeric RNA via an SPT4 dependent mechanism.

着丝粒是一种组装在着丝粒上的多蛋白复合物，对于细胞分裂过程中染色体的分离至关重要。着丝粒功能的缺陷会导致染色体不稳定和非整倍体——这是癌细胞的标志。着丝粒功能由调节蛋白的募集控制，其中许多已被记录，但它们的功能通常仍未表征，许多尚未确定。为了确定着丝粒调节的候选者，我们在出芽酵母中使用了蛋白质组范围内的蛋白质关联策略，并检测到许多参与翻译后修饰的蛋白质，例如激酶、磷酸酶和组蛋白修饰剂。我们专注于 Polo 样激酶 Cdc5，并询问哪些细胞成分对组成型 Cdc5 定位敏感。着丝粒对组成型 Cdc5 激酶活性特别敏感。将 Cdc5 靶向不同的着丝粒亚复合体产生不同的表型，与着丝粒的多种不同功能一致。我们表明，将 Cdc5 靶向内部着丝粒，组成性着丝粒相关网络 (CCAN)，通过以下方式增加着丝粒 RNA 的水平SPT4依赖机制。