CIA5, also named CCM1 (locus Cre02.g096300), is a 699-residue nuclear zinc-binding regulatory protein of Chlamydomonas reinhardtii that acts as the master regulator of acclimation to limiting inorganic carbon. Its N-terminal region carries two zinc-binding sites, a C2H2-type zinc finger (around residues 35-70) and a second Cys/His site (around residues 72-101), each of which binds one zinc ion; the rest of the protein is largely disordered, with glutamine-, alanine- and glycine-rich low-complexity tracts reminiscent of transcriptional activation domains. CIA5 mRNA and protein are present at constant levels in high and low CO2, and the protein resides in the nucleus under both conditions, so its activity is thought to be switched post-translationally when CO2 becomes limiting. Loss of CIA5 (the cia5 point mutant, His54Tyr in the first zinc site, or the ccm1 insertion mutant C16) abolishes active inorganic-carbon uptake, pyrenoid development and induction of essentially all low-CO2-inducible genes, including the bicarbonate transporters HLA3 and LCIA, the bestrophins, LCIB/LCIC, carbonic anhydrases CAH1 and CAH3, the Myb transcription factor LCR1 and the photoprotective LHCSR3, and RNA-seq shows that CIA5 affects the expression of about a quarter of all genes; the cells grow only with elevated CO2. Removing the 54 C-terminal residues yields constitutive expression of CO2-responsive genes, indicating that the C-terminus restrains its activity under high CO2. CIA5 forms a 290-580 kDa complex and binds constitutively to the nuclear CobW/WW-domain metallochaperone-like GTPase CBP1 (ZNG3), which represses a subset of CIA5-dependent genes under high CO2; under zinc deficiency CIA5 and CBP1/ZNG3 are degraded by the proteasome, preventing induction of the zinc-demanding carbon-concentrating mechanism. Whether CIA5 binds DNA itself in vivo is unresolved: it was long thought not to, but recent in vitro work reports specific binding of purified CIA5 to a GC-rich promoter motif. CIA5 appears restricted to some chlorophyte lineages, with an ortholog in Volvox carteri.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005634 nucleus | IDA PMID:41637450 A nuclear CobW/WW-domain factor represses the CO(2)-concentr... | NEW | Summary: CCM1-mGold expressed in the ccm1-1 mutant gives strong punctate nuclear fluorescence under both high and very low CO2. Nuclear localization is also reported in earlier work cited by the 2026 Plant J paper, and the Volvox ortholog conserves putative NLS/NES signals. GOA currently carries no annotation for this protein. Reason: Direct imaging evidence for nuclear localization of a functional fusion protein; consistent with its role as a transcriptional regulator and with its constitutive nuclear partner CBP1/ZNG3. Supporting Evidence: PMID:41637450 Confocal microscopy revealed strong nuclear signals in both transformants under HC and VLC conditions (Fig. 3A). PMID:41637450 Notably, the nuclear localization pattern remained unchanged between HC and VLC conditions for both proteins, showing punctate nuclear signals characteristic of transcription factor localization (Fig. 3A). PMID:42448334 CIA5 localizes to the nucleus, and cia5 mutants fail to transcriptionally induce Ci transporter genes, CAHs, and many genes encoding structural components of the CCM |
| GO:0008270 zinc ion binding | IDA PMID:18202004 Significance of zinc in a regulatory protein, CCM1, which re... | NEW | Summary: Atomic absorption spectrometry of GST fusions shows that each of the two N-terminal regions (residues 1-71 and 72-101) binds one zinc ion, and point mutations of the coordinating residues (H54Y, C77V, C80V) abolish zinc binding and CCM induction. Reason: Direct biochemical evidence of zinc binding by CIA5. Zinc binding is structural (zinc-finger fold) rather than the core regulatory activity, so it is recorded as an annotation but not as the core molecular function. Supporting Evidence: PMID:18202004 It was found that 1 mol of zinc is bound to 1 mol of amino acid regions 1-71 and 72-101 of CCM1, respectively. PMID:18202004 In the case of the site-directed mutant proteins, H54Y, C77V and C80V, the zinc-binding ability was lost. |
| GO:0071244 cellular response to carbon dioxide | IMP PMID:11287669 Ccm1, a regulatory gene controlling the induction of a carbo... | NEW | Summary: ccm1 and cia5 mutants fail to respond to CO2 limitation; they do not induce low-CO2-inducible genes, active Ci uptake or pyrenoid development, and complementation restores the response. CIA5 is the nuclear regulator through which the change in CO2 level is converted into the transcriptional acclimation program, so it carries out part of the response rather than being a downstream target of it. Reason: Mutant and complementation evidence from two independent groups plus genome-wide expression data. Participation test - CIA5 is the signal-to-transcription regulator of the response (its activity is switched by CO2 status, and a C-terminal truncation uncouples gene induction from CO2). Comparator check - transcription regulators that mediate CO2 responses in other organisms (e.g. Candida RCA1, UME6 and yeast CST6) carry GO:0071244 by IMP, so the term is used for this role. The stimulus is limiting CO2 (a decrease in CO2), which is within the scope of a response to a carbon dioxide stimulus; GO has no more specific "response to low CO2" term. Supporting Evidence: PMID:11287669 Introduction of Ccm1 into this mutant restored an active carbon transport through the CCM, development of a pyrenoid structure in the chloroplast, and induction of a set of CCM-related genes. PMID:11287669 These data indicate that Ccm1 is essential to control the induction of CCM by sensing CO2 availability in Chlamydomonas cells. PMID:11309511 CIA5 appears to be a master regulator of the carbon concentrating mechanism and is intimately involved in the signal transduction mechanism that senses and allows immediate responses to fluctuations in environmental CO2 and HCO3- concentrations. PMID:15235119 The fact that the induction of almost all low-CO2 inducible genes was impaired in the ccm1 mutant suggests that CCM1 is a master regulator of CCM through putative low-CO2 signal transduction pathways. |
| GO:0045893 positive regulation of DNA-templated transcription | IMP PMID:11309511 The Cia5 gene controls formation of the carbon concentrating... | NEW | Summary: CIA5 is a nuclear zinc-finger protein required for the transcriptional induction of CO2-responsive genes; truncation of its C-terminus causes constitutive synthesis of these mRNAs, showing that CIA5 activity itself sets their transcription. Whether it does so by binding DNA directly or as part of a larger nuclear complex is not settled, so the process term is asserted without a DNA-binding transcription factor activity. Reason: The regulatory effect is at the level of transcription (cia5 and ccm1 mutants fail to transcriptionally induce the CCM genes) and CIA5 is a constitutively nuclear protein whose own activity state (C-terminal truncation) changes target transcription, which places CIA5 among the regulators of transcription rather than as a distant upstream factor. A molecular function (DNA-binding transcription factor activity or transcription coregulator activity) is deliberately not asserted because DNA binding has been shown only in a single in vitro study not available here, and in vivo promoter occupancy has not been shown. Supporting Evidence: PMID:11309511 cia5 mutants complemented with the truncated gene exhibit constitutive synthesis of mRNAs from CO2-responsive genes in light under both high and low CO2 conditions PMID:11309511 These discoveries suggest that posttranslational changes to the C-terminal domain control the ability of CIA5 to act as an inducer and directly or indirectly control transcription of CO2-responsive genes. PMID:42448334 CIA5 localizes to the nucleus, and cia5 mutants fail to transcriptionally induce Ci transporter genes, CAHs, and many genes encoding structural components of the CCM |
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Download this section (compressed HTML)Q: Does CIA5 occupy the promoters of CCM genes in vivo (ChIP-seq or CUT&RUN), and is the GGGGCGGGG motif bound in vitro by purified CIA5 enriched at those sites? A positive answer would justify a DNA-binding transcription factor activity annotation.
Suggested experts: Spalding MH, Fukuzawa H
Q: What post-translational change activates CIA5 in low CO2, and does the CBP1/ZNG3 GTPase alter the zinc occupancy of CIA5's zinc sites?
Suggested experts: Fukuzawa H, Merchant SS
Q: Should GO add a "cellular response to low carbon dioxide" or "acclimation to CO2 limitation" term so that CO2-limitation regulators such as CIA5 and LCR1 can be annotated more specifically?
Experiment: Perform ChIP-seq on endogenous CIA5-HA knock-in lines under high and very low CO2, test motif enrichment, and use promoter reporters with the GC-rich motif mutated in a cia5 complemented background.
Hypothesis: CIA5 is a sequence-specific DNA-binding transcriptional activator of CCM genes.
Experiment: Measure zinc stoichiometry of CIA5 purified from wild type and cbp1/zng3 mutants under high and low CO2, and test whether zinc-site mutants that retain zinc constitutively bypass CBP1-mediated repression.
Hypothesis: Zinc delivery or removal by CBP1/ZNG3 toggles CIA5 activity.
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