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bioRxiv · 10.1101/2025.08.16.670667

CIA5 INTERACTS WITH THE ZINC CHAPERONE ZNG3 TO BALANCECARBON AND ZINC METABOLISM

Kusi-Appiah, G.·Schmollinger, S.·Mamo, A.·Stainbrook, S. C.·O'Halloran, T. V.·Strenkert, D.
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Abstract

Carbon and zinc (Zn) metabolism are intrinsically connected in phototrophs, as crucial components involved in CO2 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 CO2 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 CO2 environments. This increase requires the presence of Cia5, a major regulator of the CCM in Chlamydomonas. How Cia5 regulates expression of thousands of low CO2-inducible genes remains enigmatic, its transcript and protein abundance is unchanged in different CO2 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 CO2 environments. Surprisingly, zng3 mutants do not phenocopy cia5 mutants and grow well in low CO2 conditions. Instead, zng3 mutants are unable to grow like wildtype if excess carbon is available in the form of high CO2 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 CO2 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 CO2, 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 CO2 metabolism, affecting intracellular Zn trafficking and modulate the CO2 response.

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Kusi-Appiah, G., Schmollinger, S., Mamo, A., Stainbrook, S. C., O'Halloran, T. V., Strenkert, D.. 2025-08-20. CIA5 INTERACTS WITH THE ZINC CHAPERONE ZNG3 TO BALANCECARBON AND ZINC METABOLISM. https://doi.org/10.1101/2025.08.16.670667

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