BST1 (bestrophin-like protein 1; locus Cre16.g662600) is a nuclear-encoded, chloroplast-targeted multi-pass membrane protein of the bestrophin/YneE/VCCN family (Pfam Bestrophin_2) in the green alga Chlamydomonas reinhardtii. It is one of three closely related paralogs (BST1, BST2/Cre16.g663400 and BST3/Cre16.g663450) 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 places BST1 in the pentameric bestrophin channel fold, with a positively charged selectivity pore consistent with an anion channel. BST1 is induced when cells are moved to air-level CO2, and this induction depends on the CCM master regulator CIA5; it is expressed at lower levels than BST2 and BST3. Fluorescently tagged BST1 lies in the thylakoid membranes of the chloroplast and is enriched at the periphery of the pyrenoid. In interactome studies BST1 associates with its paralogs and with the stromal LCIB/LCIC complex. Knockdown of all three paralogs together (single BST3 loss has little effect) impairs growth at limiting CO2, lowers the cell's affinity for inorganic carbon and reduces inorganic carbon accumulation, especially at alkaline pH where bicarbonate dominates. 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 yet been measured directly for BST1-3. Their effect on thylakoid proton-motive force is opposite to that of Arabidopsis VCCN1, which argues against a VCCN1-like chloride-counterion role.
| 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. BST1 shares the bestrophin fold and an anion-selective pore (homology model), so anion channel activity is a sound inference. However, neither chloride selectivity nor voltage gating has been tested for BST1, and the Chlamydomonas BST1-3 paralogs 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: The IBA correctly places BST1 in an anion-channel clade, but the chloride- and voltage-specific child term is a VCCN1-specific property that the authors of the only BST1 functional study argue against for BST1-3. Generalizing to monoatomic anion channel activity keeps the well-founded family inference without asserting the Cl-/voltage specifics. A bicarbonate-specific term (GO:0160133 bicarbonate channel activity / GO:0015106) 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. BST1 is inside the bestrophin anion-channel clade, so anion channel activity transfers, but the 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. BST1 has a predicted chloroplast transit peptide and Venus-tagged BST1 localizes to chloroplast thylakoid membranes, not the plasma membrane. Reason: Contradicted by direct localization of BST1 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 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 the thylakoid proton-motive force by Cl- counter-ion flux. BST1-3 knockdown in Chlamydomonas causes only a small pmf change in the opposite direction to vccn1, with unchanged Fv/Fm and no high-light sensitivity; the BST1-3 phenotype is a CO2-concentrating-mechanism defect. Reason: A light-reaction regulatory role is the VCCN1-specific function; the BST1-3 data indicate divergence from it, and BST1 acts in inorganic-carbon delivery downstream of, not within, the light reactions. Not demonstrably wrong (BST1-3 do modestly affect pmf), but it overstates what is known for BST1. 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 localization of Venus-tagged BST1 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 BST1. 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 BST1 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-3xFLAG-tagged BST1 (PSAD promoter) co-localizes with chlorophyll autofluorescence in thylakoid membranes and is concentrated near the pyrenoid. Reason: Direct localization evidence for the more specific chloroplast thylakoid term; this is where BST1 is proposed to act. Supporting Evidence: PMID:31391312 All 3 BST-like proteins localized to the thylakoid membranes of the chloroplast PMID:31391312 Thus, BST1, BST2, and BST3 are thylakoid localized anion transporters enriched at the pyrenoid periphery |
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Download this section (compressed HTML)Q: Do BST1, BST2 and BST3 conduct bicarbonate, chloride, or both, and is BST1 a functional channel on its own or only in hetero-oligomers with BST2/BST3 (they interact directly)? Until this is measured, should GO:0015106 / GO:0160133 bicarbonate channel activity be asserted for BST1-3 at all?
Suggested experts: Moroney JV, Mackinder LCM
Q: Is the low-CO2 Ci-uptake defect of bsti knockdowns a direct consequence of lost thylakoid HCO3- flux, or partly indirect through the altered thylakoid proton-motive force?
Q: Does BST1 contribute anything distinct from BST2/BST3 given its lower expression and divergent C terminus?
Experiment: Express BST1 (with and without BST2/BST3, 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: BST1 (alone or with BST2/BST3) forms a bicarbonate-permeable anion channel.
Type: electrophysiology
Experiment: Generate bst1 single and bst1 bst2 bst3 triple CRISPR knockouts (rather than RNAi) and test growth, K1/2(Ci) and 14C-Ci accumulation, with complementation by tagged BST1 at native expression levels.
Hypothesis: BST1 is individually required for the CCM under some condition.
Type: genetic knockout and complementation
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