BST2 (bestrophin-like protein 2; locus Cre16.g663400) 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 and BST3/Cre16.g663450; more than 80% identical to one another) clustered in a ~130 kb region of chromosome 16. Older literature used the name LCI11 for BST2 and/or BST3. 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. BST2 transcripts rise within hours of transfer to air-level CO2; this induction is reduced, but less completely than for BST1 and BST3, in mutants lacking the CCM master regulator CIA5. BST2 is expressed more highly than BST1. Fluorescently tagged BST2 lies in the thylakoid membranes of the chloroplast, extends into the pyrenoid thylakoid tubules, and is concentrated near the pyrenoid periphery. In an affinity-purification interactome, BST2 was recovered as an interactor of both BST1 and BST3. RNAi lines in which all three paralogs are knocked down together grow poorly at limiting CO2, have lower affinity for inorganic carbon and accumulate less inorganic carbon, especially at alkaline pH where bicarbonate dominates; loss of BST3 alone has little effect, 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. No BST2-specific mutant has been described, and 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. BST2 shares the bestrophin fold and the anion-selective pore residues of BST1-3 (homology model), so anion channel activity is a sound inference. Neither chloride selectivity nor voltage gating has been tested for BST2, 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 paralog BST1. The IBA correctly places BST2 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 (GO:0160133 / 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. BST2 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. BST2 has a predicted chloroplast transit peptide and Venus-tagged BST2 localizes to chloroplast thylakoid membranes, not the plasma membrane. Reason: Contradicted by direct localization of BST2 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 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 is a CO2-concentrating-mechanism defect. Nothing BST2-specific bears on the light reactions. Reason: A light-reaction regulatory role is the VCCN1-specific function; the BST1-3 data indicate divergence from it, and BST2 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 BST2. Consistent with the BST1 review. 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 BST2 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 BST2. 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 BST2 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 BST2 (PSAD promoter) co-localizes with chlorophyll autofluorescence in thylakoid membranes, extends into the pyrenoid tubules and is concentrated near the pyrenoid. Reason: Direct localization evidence for BST2 itself supporting the more specific chloroplast thylakoid term; this is where BST2 is proposed to act. Caveat that the fusion was overexpressed (only BST3 was also checked under its native promoter). 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 |
Loading supporting content…
Download this section (compressed HTML)Q: Do BST1, BST2 and BST3 form homo- or hetero-pentamers in vivo, and does BST2 conduct bicarbonate, chloride, or both? Until transport is measured, should GO:0015106 / GO:0160133 bicarbonate channel activity be asserted for any of BST1-3?
Suggested experts: Moroney JV, Mackinder LCM
Q: Why is BST2 induction only partially CIA5-dependent, unlike BST1 and BST3, and does BST2 have a basal role at high CO2?
Q: Given >80% identity among BST1-3, which peptides in the interactome uniquely identified Cre16.g663400, and which studies using the name LCI11 refer to BST2 rather than BST3?
Experiment: Express BST2 (with and without BST1/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: BST2 (alone or with BST1/BST3) forms a bicarbonate-permeable anion channel.
Type: electrophysiology
Experiment: Generate bst2 single, bst2 bst3 double and bst1 bst2 bst3 triple CRISPR knockouts and test growth at low CO2, K1/2(Ci) and 14C-Ci accumulation, with complementation by tagged BST2 at native expression levels.
Hypothesis: BST2 is individually or partially redundantly required for the CCM.
Type: genetic knockout and complementation
Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)