{"doi":"10.1101/2025.08.16.670667","title":"CIA5 INTERACTS WITH THE ZINC CHAPERONE ZNG3 TO BALANCE CARBON AND ZINC METABOLISM","abstract":"ABSTRACT 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.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":558823,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9651,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":271910,"name":"Stefan Schmollinger","orcid":"0000-0002-7487-8014","position":1,"is_corresponding":false},{"id":1460060,"name":"Andrew Mamo","orcid":null,"position":2,"is_corresponding":false},{"id":823538,"name":"Sarah C. Stainbrook","orcid":"0000-0002-0124-8230","position":3,"is_corresponding":false},{"id":361448,"name":"Thomas V. O’Halloran","orcid":"0000-0001-8732-5059","position":4,"is_corresponding":false},{"id":280600,"name":"Daniela Strenkert","orcid":"0000-0002-3420-1332","position":5,"is_corresponding":false},{"id":1459626,"name":"George Kusi-Appiah","orcid":"0000-0001-8061-9265","position":0,"is_corresponding":true}],"reference_count":72,"raw_metadata":null,"created_at":"2026-07-19T02:55:30.312295Z","pmid":"40894702","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}