LCIA (Low-CO2-inducible protein A, also called NAR1.2; locus Cre06.g309000) is a nuclear-encoded member of the formate/nitrite transporter (FNT) family of anion channels in the green alga Chlamydomonas reinhardtii. The 336-residue protein carries an N-terminal chloroplast transit peptide followed by an FNT-type transmembrane domain, and it resides in the chloroplast envelope, as shown by immunofluorescence, envelope membrane fractionation and fluorescent-protein fusions. Its cryo-EM structure shows the pentameric FNT channel fold with a selectivity filter in which Lys220, together with Ala117 and Val267, sets preference for bicarbonate. LCIA moves bicarbonate across membranes: it increases bicarbonate uptake and gives HCO3- and NO2- currents in Xenopus oocytes, and it restores growth in air of an Escherichia coli mutant lacking carbonic anhydrases and of an Arabidopsis mutant lacking the plastid carbonic anhydrase betaCA5. Unlike its nitrogen-regulated NAR1 paralogs, LCIA is induced strongly at low CO2 under the control of the regulator CIA5/CCM1, and its accumulation also depends on the chloroplast Ca2+-binding protein CAS. In the algal CO2-concentrating mechanism it imports cytosolic bicarbonate into the chloroplast stroma, acting together with the plasma membrane ABC transporter HLA3. Insertion mutants have reduced inorganic carbon affinity, especially at alkaline pH where bicarbonate dominates; loss of both LCIA and LCIB prevents survival at very low CO2; and co-overexpression of LCIA with HLA3 raises inorganic carbon affinity. Loss of LCIA also lowers HLA3 mRNA levels.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005886 plasma membrane | IBA GO_REF:0000033 | MODIFY | Summary: Propagated from bacterial FNT channels (E. coli FocA, NirC, FocB), which sit in the bacterial plasma membrane. LCIA has an N-terminal chloroplast transit peptide and is found in the chloroplast envelope by immunofluorescence, envelope membrane fractionation and fluorescent fusions in Chlamydomonas, tobacco and Arabidopsis. It is not at the plasma membrane of the alga; plasma membrane bicarbonate uptake is done by HLA3. Reason: The membrane-channel nature inherited from the bacterial ancestor is correct, but the location does not transfer to this eukaryotic member. Target-specific evidence (a transit peptide that is needed for envelope targeting, and direct localization) places LCIA in the chloroplast envelope. Replace with GO:0009941 chloroplast envelope. The plasma membrane signal seen in Xenopus oocytes (PMID:26538195) used mature LCIA lacking its transit peptide and is an artefact of the heterologous assay. Propagation Review Root cause: PROPAGATION BAD Failure modes: COMPARTMENT OR COMPLEX MISMATCH Sources checked: PANTHER:PTN000775604 · PTN000775604 SUPPORTS SOURCE BUT NOT TARGET Node seeded by bacterial FNT channels that are in the bacterial plasma membrane. The eukaryotic NAR1/LCIA branch gained a chloroplast transit peptide and acts in the chloroplast envelope, so the location does not transfer to LCIA. Proposed replacements: chloroplast envelope Supporting Evidence: PMID:26015566 LCIA was highly enriched in the CE fraction, where CE protein CCP1 (30) was also enriched. PMID:26538195 LCIA: Venus was confined to the chloroplast envelope PMID:16905358 NAR1.1, NAR1.2, and NAR1.5 contain putative chloroplast transit peptides |
| GO:0015499 formate transmembrane transporter activity | IBA GO_REF:0000033 | MODIFY | Summary: Propagated from the E. coli formate channels FocA and FocB. No study reports formate transport by LCIA. Its tested substrates are bicarbonate (Xenopus oocyte currents and 14C uptake, complementation of carbonic anhydrase-deficient E. coli and Arabidopsis) and nitrite (oocyte currents). The LciA structure shows a selectivity filter tuned to bicarbonate by Lys220, Ala117 and Val267. Reason: The family-level transmembrane anion transport activity is right, but the substrate is wrong for this member. FNT channels have diverged in substrate (formate, nitrite, hydrosulfide), and the bicarbonate selectivity of LciA is a derived property that bacterial NirC lacks until engineered. With no target evidence for formate and direct evidence for bicarbonate, replace with GO:0015106 bicarbonate transmembrane transporter activity (the term the CCM module uses). Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN000775620 · PTN000775620 SUPPORTS SOURCE BUT NOT TARGET Node seeded by E. coli formate channels FocA and FocB. The FNT family has diverged in substrate, and LCIA has a bicarbonate-selective filter with no evidence of formate transport. Proposed replacements: bicarbonate transmembrane transporter activity Supporting Evidence: PMID:16905358 The electrophysiological response to HCO3- and NO2- provides evidence that NAR1.2 is involved in both HCO3- and NO2- transport. PMID:36987927 To our knowledge, this is the first direct evidence that LCIA moves HCO3- across membranes in bacteria and plants. PMID:41507353 We found that bicarbonate selectivity is governed by electrostatic coordination mediated by Lys220 and steric constraint imposed by Ala117 and Val267 within the selectivity filter. PMID:41507353 we successfully engineered the bacterial FNT family nitrite channel NirC through site-directed mutagenesis to gain bicarbonate transport activity |
| GO:0015724 formate transport | IBA GO_REF:0000033 | MODIFY | Summary: Process counterpart of the formate transporter IBA, from the same PANTHER node. LCIA has no reported role in formate transport; in the alga it moves bicarbonate from the cytosol into the chloroplast stroma as part of the CO2-concentrating mechanism. Reason: Same reasoning as for the formate transporter activity row. LCIA itself carries the bicarbonate across the envelope, so it does the work of bicarbonate transport. Mutant and overexpression data in Chlamydomonas, together with heterologous transport assays, support GO:0015701 bicarbonate transport. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN000775620 · PTN000775620 SUPPORTS SOURCE BUT NOT TARGET Same node as the formate transporter activity row; the formate substrate does not transfer to the bicarbonate-selective LCIA. Proposed replacements: bicarbonate transport Supporting Evidence: PMID:25336519 LCIB appears to function in a CO2 uptake system, whereas LCIA appears to be associated with a HCO3(-) transport system. PMID:26015566 Insertion mutants of HLA3 and/or LCIA showed decreased Ci affinities/accumulation, especially in alkaline conditions where HCO3 (-) is the predominant form of Ci. PMID:26538195 Oocytes transformed with mLCIA: GFP or HLA3: GFP accumulated 2.0‐ and 2.7‐fold more 14C than water‐injected controls, respectively |
| GO:0016020 membrane | IEA GO_REF:0000120 | ACCEPT | Summary: LCIA is a multi-pass membrane protein (six predicted transmembrane helices, FNT fold confirmed by cryo-EM) in the chloroplast envelope. Reason: Correct but general. The more informative location, chloroplast envelope, is captured through the MODIFY of the plasma membrane row and in core_functions. Supporting Evidence: PMID:26015566 we concluded that HLA3 and LCIA were localized to the PM and CE, respectively. |
| GO:0022857 transmembrane transporter activity | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the formate/nitrite transporter domain. LCIA does move anions (bicarbonate and nitrite) across membranes. Reason: Correct at the family level and an ancestor of the more specific bicarbonate transmembrane transporter activity supported by direct evidence. A broad IEA mapping is acceptable. Supporting Evidence: PMID:36987927 To our knowledge, this is the first direct evidence that LCIA moves HCO3- across membranes in bacteria and plants. |
| GO:0055085 transmembrane transport | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping. LCIA carries bicarbonate across the chloroplast envelope. Reason: Correct, though general; bicarbonate transport (GO:0015701) is the more specific process. Supporting Evidence: PMID:19321421 additional evidence supporting a role for LCIA in chloroplast envelope HCO(3)(-) transport |
Loading supporting content…
Download this section (compressed HTML)Q: Does LCIA conduct bicarbonate by passive channel-type permeation (GO:0160133 bicarbonate channel activity), as the FNT fold and the 2026 structure suggest, and if so how is net import into the stroma driven against a stromal pool that is kept alkaline in the light?
Q: Which envelope membrane does native LCIA occupy in Chlamydomonas? The inner envelope is expected and was reported for transgenic tobacco, but has not been shown in the alga.
Q: Is nitrite transport by LCIA (seen in oocytes) physiologically relevant, given that LCIA is regulated by CO2 and not by nitrogen?
Q: How does loss of LCIA lower HLA3 mRNA, and is this retrograde signal linked to the CAS-dependent Ca2+ signal from the pyrenoid?
Experiment: Reconstitute purified LciA in proteoliposomes or planar bilayers and measure HCO3- flux and single-channel currents, testing dependence on membrane potential and pH gradient, and the effect of the selectivity-filter mutants (K220, A117, V267) and the activity-enhancing K136A/A114F variant.
Hypothesis: LCIA is a passive bicarbonate channel rather than an active transporter.
Type: biophysics / electrophysiology
Experiment: Express K136A/A114F LciA in an lcia insertion mutant (and in an lcia lcib background) and measure Ci affinity at pH 9, internal Ci pool size and growth at very low CO2.
Hypothesis: The activity-enhanced LciA variants improve the CO2-concentrating mechanism in vivo.
Type: genetic complementation and physiology
Experiment: Separate inner and outer envelope membranes from low-CO2 Chlamydomonas and immunoblot for LCIA alongside inner (e.g. TIC) and outer (e.g. TOC) envelope markers, or use immunogold electron microscopy.
Hypothesis: Native LCIA resides in the inner chloroplast envelope.
Type: cell fractionation and imaging
Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)