Modulation of CHT7 complexes during light/dark- and nitrogen-mediated life-cycle transitions of Chlamydomonas

The Chlamdyomonas (Chlamydomonas reinhardtii) Compromised Hydrolysis of Triacylglycerols 7 (CHT7) protein has been previously implicated in the regulation of DNA metabolism and cell cycle-related gene expression during nitrogen (N) deprivation, and its predicted protein-interaction domains are necessary for function. Here, we examined impacts of the cht7 mutation during the cell division cycle under nutrient deficiency in light-dark synchronized cultures. We explored the potential mechanisms affecting CHT7 complex activities during the cell cycle and N starvation, with a focus on the possible interaction between CHT7 and the Chlamydomonas retinoblastoma tumor suppressor (RB) protein homolog, MAT3. Notably, the absence of CHT7 did not negatively impact the synchrony of cell division and cell cycle progression during diel growth. Although the majority of CHT7 and MAT3/RB proteins were observed in separate complexes by blue native-PAGE (BN-PAGE), the two proteins coimmunoprecipitated both during synchronized growth and following N deprivation, suggesting the presence of low abundance sub-complexes containing CHT7 and MAT3/RB. Furthermore, we observed several phosphorylated isoforms of CHT7 under these conditions. To test the potential role of phosphorylation on the structure and function of CHT7, we performed site directed mutagenesis of previously identified phosphorylated amino acids within CHT7. These phosphorylated residues were dispensable for CHT7 function, but phosphorylated variants of CHT7 persisted, indicating that yet unidentified residues within CHT7 are also likely phosphorylated. Based on the interaction of CHT7 and MAT3/RB, we postulate the presence of a low-abundance or transient regulatory complex in Chlamydomonas which may be similar to DREAM-like complexes in other organisms.