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 ₂ assimilation, like carbonic anhydrases, are highly abundant Zn proteins. Utilizing these and other proteins, the eukaryotic green algae Chlamydomonas reinhardtii can maintain phototrophic growth in low CO ₂ 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 ₂ 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 ₂ -inducible genes remains enigmatic, its transcript and protein abundance is unchanged in different CO ₂ 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 CIA5 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 ₂ environments. Surprisingly, zng3 mutants do not phenocopy cia5 mutants and grow well in low CO ₂ conditions. Instead, zng3 mutants are unable to grow like wildtype if excess carbon is available in the form of high CO ₂ or acetate. Transcriptomics of wildtype and zng3 mutants grown with different carbon sources revealed that transcriptional induction of the majority of genes involved in the CCM is maintained in low CO ₂ grown zng3 mutants, while the degree of induction in a subset of LCI genes is reduced ( HLA3 , CAH4 and CAH5 ). Genes encoding proteins involved in plastid quality control were induced in zng3 mutants grown on acetate and high CO ₂ , as well as other, related metallochaperones. We hypothesize that Zn trafficking towards the plastid is mis regulated in zng3 mutants resulting in protein mis-metalation and unfolding. Taken together, we propose that ZNG3 and Cia5 coordinate Zn and CO ₂ metabolism, affecting intracellular Zn trafficking and modulate the CO ₂ response.