ChELSI Institute, Department of Chemical and Biological Engineering, University of Sheffield, Sheffield, S1 3JD, UK.
Department of Molecular Biology and Biotechnology, University of Sheffield, S10 2TN, Sheffield, UK.
Sci Rep. 2019 Apr 18;9(1):6258. doi: 10.1038/s41598-019-42530-1.
The chromosome complement of the human fungal pathogen Candida albicans is unusually unstable, suggesting that the process of nuclear division is error prone. The Cdc14 phosphatase plays a key role in organising the intricate choreography of mitosis and cell division. In order to understand the role of Cdc14 in C. albicans we used quantitative proteomics to identify proteins that physically interact with Cdc14. To distinguish genuine Cdc14-interactors from proteins that bound non-specifically to the affinity matrix, we used a substrate trapping mutant combined with mass spectrometry analysis using Stable Isotope Labelling with Amino Acids in Cell Culture (SILAC). The results identified 126 proteins that interact with Cdc14 of which 80% have not previously been identified as Cdc14 interactors in C. albicans or S. cerevisiae. In this set, 55 proteins are known from previous research in S. cerevisiae and S. pombe to play roles in the cell cycle, regulating the attachment of the mitotic spindle to kinetochores, mitotic exit, cytokinesis, licensing of DNA replication by re-activating pre-replication complexes, and DNA repair. Five Cdc14-interacting proteins with previously unknown functions localised to the Spindle Pole Bodies (SPBs). Thus, we have greatly increased the number of proteins that physically interact with Cdc14 in C. albicans.
人类真菌病原体白色念珠菌的染色体组非常不稳定,这表明核分裂过程容易出错。Cdc14 磷酸酶在组织有丝分裂和细胞分裂的复杂舞蹈中起着关键作用。为了了解 Cdc14 在白色念珠菌中的作用,我们使用定量蛋白质组学来鉴定与 Cdc14 物理相互作用的蛋白质。为了将与 Cdc14 特异性结合的蛋白与真正的 Cdc14 相互作用蛋白区分开来,我们使用了一种底物捕获突变体,并结合使用了 Stable Isotope Labelling with Amino Acids in Cell Culture (SILAC) 的质谱分析。结果鉴定出 126 种与 Cdc14 相互作用的蛋白质,其中 80%以前未被鉴定为白色念珠菌或酿酒酵母中的 Cdc14 相互作用蛋白。在这一组中,有 55 种蛋白质以前在酿酒酵母和粗糙脉孢菌的研究中被发现参与细胞周期,调节有丝分裂纺锤体与动粒的附着、有丝分裂退出、胞质分裂、通过重新激活预复制复合物来许可 DNA 复制以及 DNA 修复。五个具有未知功能的与 Cdc14 相互作用的蛋白定位于纺锤体极体(SPB)。因此,我们大大增加了与 Cdc14 在白色念珠菌中物理相互作用的蛋白质数量。