CIA5 and its interacting metal-binding GTPase ZNG3 are degraded by the proteasome in Zn deficiency

Carbon and zinc (Zn) metabolism are tightly connected in phototrophs, as carbonic anhydrases critical for CO₂ assimilation are highly abundant Zn enzymes. The eukaryotic green alga Chlamydomonas (Chlamydomonas reinhardtii) maintains efficient phototrophic growth in ambient or low CO₂ environments by establishing a carbon concentrating mechanism (CCM). We show that Chlamydomonas raises its cellular Zn quota to accommodate higher Zn demand under low CO₂ conditions, an adjustment that is dependent on CIA5, a major regulator of the CCM. We demonstrate that CIA5 is constitutively expressed regardless of Zn supply, but its encoded protein undergoes proteasomal degradation under Zn deficiency, providing a checkpoint that prevents CCM induction when Zn cofactors are unavailable. We identified a COBW domain-containing GTPase, ZNG3, as a constitutive interacting partner of CIA5. As CIA5, ZNG3 does not accumulate in low Zn and is degraded by the proteasome. Unlike cia5 mutants, zng3 mutants grow like wild-type without CO₂ supplementation but exhibit a growth defect when grown in the presence of high CO₂ or acetate. Transcriptome sequencing revealed that expression of genes encoding central components of the CCM is mostly unchanged in zng3 mutants, while a subset of genes induced in Zn deficiency, including ZRT2 encoding for a Zn transporter, is increasingly expressed. Under low CO₂ conditions, cia5 zng3 double mutants exhibit a more severe phenotype than cia5 single mutants. This result points to a condition-dependent genetic interaction between CIA5 and ZNG3, suggesting both proteins provide distinct but partially overlapping contributions to fitness depending on carbon availability.