LCIB (Low-CO2-inducible protein B) is a nuclear-encoded, chloroplast-targeted soluble protein of about 48 kDa in the green alga Chlamydomonas reinhardtii, strongly induced when CO2 is limiting. It is the gene product affected in the classical air-dier mutants pmp1 and ad1, which cannot grow at air levels of CO2 but grow at high or very low CO2 and fail to accumulate inorganic carbon at air CO2. LCIB belongs to a small algal and bacterial family (with LCIC, LCID and LCIE in Chlamydomonas) whose crystal structures reveal a beta-carbonic anhydrase fold with a zinc ion bound by two cysteines and a histidine. Some family members from diatoms and bacteria are active carbonic anhydrases. Purified Chlamydomonas LCIB, and the native LCIB-LCIC complex, showed no activity in vitro, but LCIB restores growth of yeast and Arabidopsis carbonic anhydrase knockout mutants and raises carbonic anhydrase activity when expressed in the tobacco chloroplast stroma, indicating that it can hydrate CO2 to bicarbonate and that its activity may be tightly regulated. In vivo LCIB forms a large hetero-oligomeric complex (about 350-440 kDa) with its paralog LCIC. The complex is dispersed in the chloroplast stroma at high CO2 and moves to form a ring around the pyrenoid, outside the starch sheath, when CO2 falls; this relocation needs LCIC and an intact starch sheath. Genetic suppressor analysis places LCIB downstream of the thylakoid lumen carbonic anhydrase CAH3, and the favoured model is that the stromal LCIB-LCIC complex converts CO2 that escapes the pyrenoid, or enters the chloroplast, into bicarbonate, keeping inorganic carbon trapped in the stroma for the CO2-concentrating mechanism.
Definition: A soluble hetero-oligomeric protein complex of the chloroplast stroma of green algae, consisting of the beta-carbonic anhydrase-fold paralogs LCIB and LCIC, that relocates to the periphery of the pyrenoid under limiting CO2 and is proposed to hydrate CO2 to bicarbonate.
Justification: LCIB and LCIC interact in yeast and in vivo and co-purify as a 350-440 kDa complex; LCIC is unstable without LCIB and LCIB relocation requires LCIC. No GO cellular component term exists for this complex, so in_complex cannot be filled.
Parent term: protein-containing complex
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004089 carbonate dehydratase activity | IGI PMID:36856938 LCIB functions as a carbonic anhydrase: evidence from yeast ... | NEW | Summary: LCIB has a beta-carbonic anhydrase fold with a conserved zinc site, complements carbonic anhydrase knockout mutants of yeast (NCE103) and Arabidopsis (betaCA5), and raises total CA activity when expressed in the tobacco chloroplast stroma. Purified Chlamydomonas LCIB, LCIC and the LCIB-LCIC complex were inactive in vitro (Jin et al. 2016, full text), so the activity appears to be conditional or tightly regulated. Reason: No GOA annotations exist for Q75NZ2. GO:0004089 (catalysis of hydrogencarbonate + H+ = CO2 + H2O) is the activity proposed for LCIB in the CCM. The evidence is two independent heterologous complementation assays (PMID:36856938), increased CA activity in transgenic tobacco (PMID:42328090), and a beta-CA active-site zinc site in the crystal structure (PMID:27911826). Because LCIB alone complements, the activity is assigned to LCIB rather than only as a contribution to the LCIB-LCIC complex. Users should note that direct kinetic measurement on purified Chlamydomonas protein was negative in Jin et al. 2016; the CA-active family members in that study were diatom and bacterial homologs. Supporting Evidence: PMID:36856938 We provide evidence that LCIB is an active CA using a Saccharomyces cerevisiae CA knockout mutant (βNCE103) and an Arabidopsis thaliana Ξ²CA5 knockout mutant (Ξ²ca5). PMID:36856938 We show that different truncated versions of the LCIB protein complement βNCE103, while the full length LCIB protein complements Ξ²ca5 plants, so that both the yeast and plant mutants can grow in low CO2 conditions. PMID:42328090 LCIB was exclusively localized in the stroma and remained biologically active, leading to a ~3-fold increase in total carbonic anhydrase activity and a lower apparent CO2 compensation point (-10%). PMID:27911826 We determined the crystal structures of LCIB and limiting CO2-inducible C protein (LCIC) from C. reinhardtii and a CA-functional homolog from Phaeodactylum tricornutum, all of which harbor motifs bearing close resemblance to the active site of canonical Ξ²-CAs. |
| GO:0009570 chloroplast stroma | IDA PMID:34791500 CO2-dependent migration and relocation of LCIB, a pyrenoid-p... | NEW | Summary: LCIB is a soluble stromal protein. It is dispersed in the chloroplast stroma at high and low CO2 and gathers in the stroma around the pyrenoid, outside the starch sheath, at very low CO2. Reason: No GOA annotations exist for Q75NZ2. Live imaging of LCIB-Clover (PMID:34791500) and immunolocalization (PMID:20660228) place LCIB in the chloroplast stroma, and the protein has no transmembrane segment or starch-binding domain (PMID:32041908). GO:1990732 pyrenoid is not proposed, because LCIB lies outside the starch sheath and not in the Rubisco matrix. Supporting Evidence: PMID:34791500 LC-inducible protein B (LCIB), structurally characterized as carbonic anhydrase, localizes in the chloroplast stroma under CO2-supplied and LC conditions. PMID:32041908 Because LCIB and LCIC do not have a starch-binding domain and exist as soluble proteins in the chloroplast stroma |
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Download this section (compressed HTML)Q: Why are purified Chlamydomonas LCIB and the native LCIB-LCIC complex inactive in vitro while LCIB complements yeast and Arabidopsis CA knockouts? Is activity switched on by a post-translational modification, a partner such as LCIC in a defined stoichiometry, redox state, or the C-terminal region absent from the crystallized construct?
Suggested experts: Moroney JV, Mueller-Cajar O, Fukuzawa H
Q: Should GO add a biological process term for the pyrenoid-based CO2-concentrating mechanism (or a general "inorganic carbon concentrating mechanism"), so that stromal and lumenal carbonic anhydrases, bicarbonate channels and pyrenoid linkers can be annotated to a shared process?
Q: Does LCIB-LCIC act as a unidirectional ("vectorial") CO2 hydratase coupled to some energy source, or as an ordinary reversible CA whose position relative to the pyrenoid alone sets the net direction of flux?
Experiment: Measure 18O-exchange (MIMS) activity of full-length LCIB, LCIB with LCIC co-expressed at native stoichiometry, and complex purified from very-low-CO2 cells under varied redox, pH and Zn conditions; test zinc-ligand mutants (C120A, H180A, C204A) for loss of yeast nce103 complementation.
Hypothesis: Chlamydomonas LCIB has intrinsic CO2 hydration activity that is masked under standard in vitro conditions.
Type: biochemistry and complementation
Experiment: Complement ad1 or pmp1 with wild-type LCIB versus zinc-site mutants, and with a heterologous active beta-CA (for example PtLCIB4) targeted to the stroma and to the pyrenoid periphery; score growth at air CO2 and Ci accumulation.
Hypothesis: CA activity, rather than a structural role, explains the lcib air-dier phenotype.
Type: genetic complementation and physiology
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