Cosma Maria-Alexa, Curtis Natalie L, Pain Charlotte, Kriechbaumer Verena, Bolanos-Garcia Victor M
Department of Biological and Medical Sciences, Faculty of Health and Life Sciences, Oxford Brookes University, Headington, OX, United Kingdom.
Front Physiol. 2022 Jul 25;13:938688. doi: 10.3389/fphys.2022.938688. eCollection 2022.
The Anaphase Promoting Complex (APC/C), a large cullin-RING E3-type ubiquitin ligase, constitutes the ultimate target of the Spindle Assembly Checkpoint (SAC), an intricate regulatory circuit that ensures the high fidelity of chromosome segregation in eukaryotic organisms by delaying the onset of anaphase until each chromosome is properly bi-oriented on the mitotic spindle. Cell-division cycle protein 20 homologue (CDC20) is a key regulator of APC/C function in mitosis. The formation of the APC/C complex is required for the ubiquitination and degradation of select substrates, which is necessary to maintain the mitotic state. In contrast to the roles of CDC20 in animal species, little is known about CDC20 roles in the regulation of chromosome segregation in plants. Here we address this gap in knowledge and report the expression in insect cells; the biochemical and biophysical characterisation of (AtCDC20) WD40 domain; and the nuclear and cytoplasmic distribution of full-length AtCDC20 when transiently expressed in tobacco plants. We also show that most AtCDC20 degrons share a high sequence similarity to other eukaryotes, arguing in favour of conserved degron functions in AtCDC20. However, important exceptions were noted such as the lack of a canonical MAD1 binding motif; a fully conserved RRY-box in all six AtCDC20 isoforms instead of a CRY-box motif, and low conservation of key residues known to be phosphorylated by BUB1 and PLK1 in other species to ensure a robust SAC response. Taken together, our studies provide insights into AtCDC20 structure and function and the evolution of SAC signalling in plants.
后期促进复合物（APC/C）是一种大型的cullin-RING E3型泛素连接酶，是纺锤体组装检查点（SAC）的最终靶点。纺锤体组装检查点是一个复杂的调节回路，通过延迟后期的开始，直到每条染色体在有丝分裂纺锤体上正确地双定向，从而确保真核生物中染色体分离的高保真度。细胞分裂周期蛋白20同源物（CDC20）是有丝分裂中APC/C功能的关键调节因子。APC/C复合物的形成是某些底物泛素化和降解所必需的，而这对于维持有丝分裂状态是必要的。与CDC20在动物物种中的作用不同，人们对CDC20在植物染色体分离调控中的作用知之甚少。在这里，我们填补了这一知识空白，报告了其在昆虫细胞中的表达；拟南芥CDC20（AtCDC20）WD40结构域的生化和生物物理特性；以及全长AtCDC20在烟草植物中瞬时表达时的核质分布。我们还表明，大多数AtCDC20降解子与其他真核生物具有高度的序列相似性，这支持了AtCDC20中保守的降解子功能。然而，也注意到了一些重要的例外情况，例如缺乏典型的MAD1结合基序；所有六种AtCDC20异构体中都有一个完全保守的RRY框，而不是CRY框基序；以及其他物种中已知被BUB1和PLK1磷酸化以确保强大的SAC反应的关键残基的保守性较低。综上所述，我们的研究为AtCDC20的结构和功能以及植物中SAC信号的进化提供了见解。
