BST3 (bestrophin-like protein 3; locus Cre16.g663450; called LCI11 in the 2017 CO2-concentrating-mechanism interactome) is a nuclear-encoded, chloroplast-targeted multi-pass membrane protein of the bestrophin/YneE/VCCN family in the green alga Chlamydomonas reinhardtii. It is one of three closely related paralogs (BST1/Cre16.g662600, BST2/Cre16.g663400 and BST3; more than 80% identical to one another) clustered in a ~130 kb region of chromosome 16. Its nearest land-plant relative is the Arabidopsis thylakoid voltage-dependent chloride channel VCCN1 (about 30% identity). Homology modelling of the BST1-3 group places them in the pentameric bestrophin channel fold, with a positively charged selectivity pore consistent with an anion channel. BST3 transcripts rise within two hours of transfer to air-level CO2, and BST3 expression is severely reduced in mutants lacking the CCM master regulator CIA5, as for other CCM genes. A Venus fusion expressed from the native BST3 promoter lies in the thylakoid membranes of the chloroplast, extends into the pyrenoid thylakoid tubules, and is about 1.5-fold enriched at the pyrenoid periphery. In an affinity-purification interactome, BST3 interacted with BST1 and BST2 and with the stromal LCIB/LCIC complex that sits around the pyrenoid. A CLiP insertion mutant lacking BST3 protein has only a weak growth defect and slightly lower inorganic-carbon uptake at air-level CO2, whereas RNAi lines in which all three paralogs are knocked down grow poorly at limiting CO2, have lower affinity for inorganic carbon and accumulate less of it, implying redundancy. BST1-3 are therefore proposed to be redundant thylakoid anion channels that move stromal bicarbonate into the thylakoid lumen, where carbonic anhydrase CAH3 converts it to CO2 for Rubisco in the pyrenoid. Bicarbonate (or any anion) conductance has not been measured for any of BST1-3.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005247 voltage-gated chloride channel activity | IBA GO_REF:0000033 | MODIFY | Summary: Phylogenetic transfer from Arabidopsis VCCN1, an electrophysiologically characterized voltage-dependent thylakoid Cl- channel. BST3 shares the bestrophin fold and the anion-selective pore of BST1-3 (homology model), so anion channel activity is a sound inference. Neither chloride selectivity nor voltage gating has been tested for BST3, and BST1-3 behave differently from VCCN1 in vivo (knockdown lowers rather than raises thylakoid pmf). Their physiological role is linked to inorganic carbon (proposed HCO3-) delivery to the lumen. Reason: Same reasoning as for the near-identical paralogs BST1 and BST2. The IBA correctly places BST3 in an anion-channel clade, but the chloride- and voltage-specific child term is a VCCN1-specific property that the only functional study of BST1-3 argues against. Generalizing to monoatomic anion channel activity keeps the family inference without asserting Cl-/voltage specifics. A bicarbonate-specific term would be premature until transport is measured. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: FUNCTIONAL DIVERGENCE GRANULARITY MISMATCH Sources checked: PANTHER:PTN004990962 · PTN004990962 SUPPORTS SOURCE BUT NOT TARGET Node grounded by Arabidopsis VCCN1 (AT3G61320), a bilayer-verified voltage-dependent Cl- channel. BST3 is inside the bestrophin anion-channel clade, so anion channel activity transfers, but Cl-/voltage specifics are not shown for BST1-3, whose in vivo pmf effect is opposite to VCCN1. Proposed replacements: monoatomic anion channel activity Supporting Evidence: PMID:31391312 the selective pore is positively charged, supporting the hypothesis that BST1–3 transport negatively charged ions PMID:31391312 The presence of AtVCCN1 decreases pmf in Arabidopsis, but the presence of the 3 BST proteins increases pmf in Chlamydomonas. PMID:27216227 In this study, we bring evidence that AtVCCN1 forms a voltage-dependent Cl− channel, is located in the thylakoid membrane |
| GO:0005886 plasma membrane | IEA GO_REF:0000044 | REMOVE | Summary: Electronic mapping from an ARBA-predicted UniProt "Cell membrane" subcellular location. BST3 has a predicted chloroplast transit peptide, and Venus-tagged BST3, under both the PSAD and its native promoter, localizes to chloroplast thylakoid membranes, not the plasma membrane. Reason: Contradicted by direct native-promoter localization of BST3 to thylakoid membranes and by transit-peptide prediction; the ARBA rule applies the default bestrophin (animal/bacterial plasma membrane) location to a chloroplast paralog. Supporting Evidence: PMID:31391312 All 3 BST-like proteins localized to the thylakoid membranes of the chloroplast PMID:31391312 This line showed the same localization pattern as BST3 under the constitutive PSAD promoter PMID:31391312 predicted that each BST protein had a chloroplast transit peptide and was likely to be a chloroplast membrane protein |
| GO:0019684 photosynthesis, light reaction | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: Transferred from Arabidopsis VCCN1, which fine-tunes thylakoid proton-motive force by Cl- counter-ion flux. BST1-3 triple knockdown in Chlamydomonas causes only a small pmf change in the opposite direction to vccn1; the BST1-3 phenotype, and the weak bst3 single-mutant phenotype, are CO2-concentrating-mechanism (Ci uptake) defects. Reason: A light-reaction regulatory role is the VCCN1-specific function; the BST1-3 data indicate divergence from it, and BST3 is proposed to act in inorganic-carbon delivery downstream of, not within, the light reactions. Not demonstrably wrong (BST1-3 modestly affect pmf), but it overstates what is known for BST3. Consistent with the BST1 and BST2 reviews. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN008436049 · PTN008436049 SUPPORTS SOURCE BUT NOT TARGET Process grounded by VCCN1 regulation of thylakoid pmf in Arabidopsis. Chlamydomonas BST1-3 knockdowns show the opposite small pmf change and a CCM rather than light-reaction phenotype. Supporting Evidence: PMID:31391312 suggests that the function of the BST proteins in Chlamydomonas is not the same as VCCN1 in Arabidopsis PMID:31391312 The presence of AtVCCN1 decreases pmf in Arabidopsis, but the presence of the 3 BST proteins increases pmf in Chlamydomonas. |
| GO:0042651 thylakoid membrane | IBA GO_REF:0000033 | ACCEPT | Summary: Consistent with direct native-promoter localization of Venus-tagged BST3 to the chloroplast thylakoid membranes, enriched near the pyrenoid, and with the thylakoid location of the Arabidopsis donors VCCN1/VCCN2. Reason: Correct; experimentally confirmed for BST3. The more specific chloroplast thylakoid membrane term is added below as a NEW IDA row. Supporting Evidence: PMID:31391312 All 3 BST-like proteins localized to the thylakoid membranes of the chloroplast |
| GO:1902476 chloride transmembrane transport | IEA GO_REF:0000108 | MODIFY | Summary: Inter-ontology inference from the IBA voltage-gated chloride channel activity row. Chloride transport by BST3 has not been tested, and the in vivo data suggest BST1-3 differ from the VCCN1 Cl- channel. Reason: Follows the MF row; generalize to anion transmembrane transport to match the modified anion-channel activity rather than asserting chloride specifically. Proposed replacements: monoatomic anion transmembrane transport Supporting Evidence: PMID:31391312 the selective pore is positively charged, supporting the hypothesis that BST1–3 transport negatively charged ions |
| GO:0009535 chloroplast thylakoid membrane | IDA PMID:31391312 Thylakoid localized bestrophin-like proteins are essential f... | NEW | Summary: Venus-tagged BST3 co-localizes with chlorophyll autofluorescence in thylakoid membranes, extends into the pyrenoid tubules and is enriched ~1.46-fold at the pyrenoid periphery. Uniquely among BST1-3, this was confirmed with a fusion expressed from the native BST3 promoter. Reason: Direct localization evidence for BST3 itself, at native expression level, supporting the more specific chloroplast thylakoid term; this is where BST3 is proposed to act. Supporting Evidence: PMID:31391312 All 3 BST-like proteins localized to the thylakoid membranes of the chloroplast PMID:31391312 This line showed the same localization pattern as BST3 under the constitutive PSAD promoter (Fig. 3C), and quantification of enrichment showed a 1.46-fold enrichment |
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Download this section (compressed HTML)Q: Does BST3 conduct bicarbonate, chloride, or both, and do BST1-3 form hetero-pentamers in vivo? Until transport is measured, should a bicarbonate channel term be asserted for any of BST1-3?
Suggested experts: Moroney JV, Mackinder LCM
Q: Is the BST3-LCIB/LCIC interaction direct, and does LCIB/LCIC gate or position BST3 at the pyrenoid periphery to recapture leaked CO2 as bicarbonate?
Q: Which other papers using the name LCI11 refer to BST3 (Cre16.g663450) rather than BST2 (Cre16.g663400)?
Experiment: Express BST3 (with and without BST1/BST2, and C-terminally truncated to relieve possible autoinhibition) in HEK293 cells or Xenopus oocytes, or reconstitute purified protein in planar bilayers, and measure reversal potentials and relative permeability for HCO3- versus Cl-.
Hypothesis: BST3 (alone or with BST1/BST2) forms a bicarbonate-permeable anion channel.
Type: electrophysiology
Experiment: Generate bst1 bst2 double and bst1 bst2 bst3 triple CRISPR knockouts in the bst3 CLiP background and complement with native-promoter BST3; test growth at low CO2, K1/2(Ci) and 14C-Ci accumulation.
Hypothesis: BST1-3 act redundantly, with BST3 sufficient for the CCM when the other two are absent.
Type: genetic knockout and complementation
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