DYRK protein kinases in Chlamydomonas reinhardtii: Biochemical characterization and physiological study of a DYRK mutant strain

莱茵衣藻中的 DYRK 蛋白激酶：DYRK 突变株的生化特征和生理学研究

• 批准号: 235301650
• 负责人: Dr. Miriam Schulz-Raffelt
• 金额: --
• 依托单位: Fachgebiet Molekulare Biotechnologie und Systembiologie
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2013-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/235301650?language=en
• 关键词: DYRK; protein; kinases; Chlamydomonas; reinhardtii

Dual-specificity tyrosine-phosphorylation regulated kinases (DYRKs) are found in all eukaryotes studied so far and share common structural and biochemical properties. DYRK members participate in several signalling pathways, which are important for cell homeostasis and development. However, the role of DYRK proteins in plants is still unknown. Recently, a DYRK mutant of Chlamydomonas (std1) was isolated in a forward genetic approach to screen for starch degradation mutants. Based on phylogenetic analysis, we discovered a new subgroup (DYRKP) that is only composed of DYRK homologs of the green lineage. Recent data indicate that DYRKP-1 (Std1) does not only play a role in starch degradation leading to a decreased initial hydrogen production, but also in the cellular stress response to nitrogen deprivation during autotrophic condition causing higher starch accumulation, photosynthetic activity and biomass production in the mutant. In this project we want to further characterize the DYRK mutant physiologically and the corresponding kinase biochemically. Does only nitrogen deprivation trigger the observed phenotype? What happens in different light intensities? We are also interested to find out if there is a link between higher starch accumulation and higher photosynthetic activity using a starchless strain. In a biochemical approach we want to generate an HA-tagged strain that will be used to localize the protein and will also be very useful for future analysis like the search for kinase substrates and interaction partners. Finally, we plan to test the functionality of the conserved autophosphorylation motif of Chlamydomonas DYRKP-1 via site-directed mutagenesis.

双特异性酪氨酸磷酸化调节激酶（DYRK）存在于迄今为止研究的所有真核生物中，并且具有共同的结构和生化特性。 DYRK 成员参与多种信号通路，这对于细胞稳态和发育非常重要。然而，DYRK 蛋白在植物中的作用仍不清楚。最近，通过正向遗传方法分离出衣藻 (std1) 的 DYRK 突变体，以筛选淀粉降解突变体。基于系统发育分析，我们发现了一个新的亚群（DYRKP），它仅由绿色谱系的DYRK同源物组成。最近的数据表明，DYRKP-1 (Std1) 不仅在淀粉降解中发挥作用，导致初始氢产量减少，而且还在自养条件下对氮剥夺的细胞应激反应中发挥作用，导致突变体中淀粉积累、光合活性和生物量产生更高。在这个项目中，我们希望进一步表征 DYRK 突变体的生理学特征和相应激酶的生化特征。只有氮剥夺才会触发观察到的表型吗？不同光照强度下会发生什么？我们还有兴趣找出使用无淀粉菌株时较高的淀粉积累和较高的光合作用活性之间是否存在联系。在生化方法中，我们希望生成一种带有 HA 标签的菌株，该菌株将用于定位蛋白质，并且对于未来的分析（例如寻找激酶底物和相互作用伙伴）也非常有用。最后，我们计划通过定点诱变测试衣藻 DYRKP-1 保守自磷酸化基序的功能。