CIA5 INTERACTS WITH THE ZINC CHAPERONE ZNG3 TO BALANCE CARBON AND ZINC METABOLISM.

Carbon and zinc (Zn) metabolism are intrinsically connected in phototrophs, as crucial components involved in CO assimilation, like carbonic anhydrases, are highly abundant Zn proteins. Utilizing these and other proteins, the eukaryotic green algae can maintain phototrophic growth in low CO environments by inducing a carbon concentrating mechanism (CCM). In this work we show that Chlamydomonas dynamically increases its Zn content to accommodate the higher intracellular Zn demand in low CO environments. This increase requires the presence of Cia5, a major regulator of the CCM in Chlamydomonas. How Cia5 regulates expression of thousands of low CO -inducible genes remains enigmatic, its transcript and protein abundance is unchanged in different CO environments, even in the presence of an additional reduced carbon source, acetate. We show here that the Cia5 protein is not present in Zn-limitation, despite transcription being unchanged. We used a CRISPR knock-in approach to express Cia5-HA from its endogenous locus and used two independent Cia5-HA expressing strains for affinity purification and identified a protein belonging to a conserved family of metal binding GTPases, ZNG3, as a constitutive interaction partner. Like Cia5, ZNG3 is constitutively expressed, co-expressed with Cia5 along the diurnal cycle and is Cia5-dependently induced in low CO environments. Surprisingly, mutants do not phenocopy mutants and grow well in low CO conditions. Instead, mutants are unable to grow like wildtype if excess carbon is available in the form of high CO or acetate. Transcriptomics of wildtype and mutants grown with different carbon sources revealed that transcriptional induction of the majority of genes involved in the CCM is maintained in low CO grown mutants, while the degree of induction in a subset of genes is reduced ( , and ). Genes encoding proteins involved in plastid quality control were induced in mutants grown on acetate and high CO , as well as other, related metallochaperones. We hypothesize that Zn trafficking towards the plastid is mis regulated in mutants resulting in protein mis-metalation and unfolding. Taken together, we propose that ZNG3 and Cia5 coordinate Zn and CO metabolism, affecting intracellular Zn trafficking and modulate the CO response.