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

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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.