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

到目前为止，在所有研究的真核生物中都发现了双特异性酪氨酸-磷酸化调节蛋白激酶(DYRKs)，它们具有共同的结构和生化特性。Dyrk成员参与了几个信号通路，这些通路对细胞的动态平衡和发育至关重要。然而，Dyrk蛋白在植物中的作用仍不清楚。最近，用正向遗传方法分离到衣藻Dyrk突变体(Std1)，用于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)，该亚群仅由绿色系的Dyrk同源物组成。最近的数据表明，DYRKP-1(Std1)不仅在淀粉降解导致初始产氢减少，而且在自养条件下细胞对氮剥夺的胁迫反应中发挥作用，导致突变体中更高的淀粉积累、光合作用活性和生物量生产。在这个项目中，我们想要进一步研究Dyrk突变体的生理特性和相应的激酶的生物化学特性。是否只有缺氮才会触发观察到的表型？在不同的光强度下会发生什么？我们还感兴趣的是，使用无淀粉菌株是否存在较高的淀粉积累和较高的光合作用活性之间的联系。在一种生化方法中，我们希望产生一种HA标记的菌株，用于定位蛋白质，并对未来的分析非常有用，如寻找激酶底物和相互作用伙伴。最后，我们计划通过定点突变来测试衣藻DYRKP-1保守的自动磷酸化基序的功能。

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.