Modulation of CHT7 Complexes during Light/Dark- and Nitrogen-Mediated Life Cycle Transitions of Chlamydomonas.

The Chlamydomonas reinhardtii Compromised Hydrolysis of Triacylglycerols7 (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 C. reinhardtii 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, the two proteins coimmunoprecipitated both during synchronized growth and following N deprivation, suggesting the presence of low abundance subcomplexes 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 C. reinhardtii that may be similar to DREAM-like complexes in other organisms.

Chlamydomonas CHT7 Is Required for an Effective Quiescent State by Regulating Nutrient-Responsive Cell Cycle Gene Expression.

COMPROMISED HYDROLYSIS OF TRIACYLGLYCEROLS7 (CHT7) in Chlamydomonas (Chlamydomonas reinhardtii) was previously shown to affect the transcription of a subset of genes during nitrogen (N)-replete growth and following N refeeding. Here, we show that an extensive derepression of genes involved in DNA metabolism and cell cycle-related processes, as well as downregulation of genes encoding oxidoreductases and nutrient transporters, occurs in the cht7 mutant during N deprivation. Cellular mutant phenotypes are consistent with the observed transcriptome misregulation, as cht7 cells fail to properly arrest growth, nuclear replication, and cell division following N deprivation. Reduction in cht7 colony formation following N refeeding is explained by its compromised viability during N deprivation and by the occurrence of abortive divisions during N refeeding. Surprisingly, the largely unstructured C-terminal half of CHT7 with predicted protein binding domains, but not the canonical CXC DNA binding domain, is essential for the ability of CHT7 to form stable complexes and reverse the cellular phenotypes and transcription levels in the cht7 mutant. Hence, although lacking the presumed DNA binding domain, CHT7 modulates the expression of cell cycle genes in response to N availability, which is essential for establishing an effective quiescent state and the coordinated resumption of growth following N refeeding.