RBMP1, also called BST4 (bestrophin-like protein 4; locus Cre06.g261750), is a nuclear-encoded, chloroplast-targeted membrane protein of about 668 residues in the green alga Chlamydomonas reinhardtii. Its N-terminal part is a bestrophin (YneE/VCCN1-like) channel domain related to the thylakoid bestrophins BST1-3 and the plant thylakoid chloride channel VCCN1. Its long, intrinsically disordered C-terminus faces the stroma and carries two Rubisco-binding motifs that bind the alpha-helices of the Rubisco small subunit. The protein is found only in the pyrenoid tubules, the thylakoid-derived membranes that cross the Rubisco matrix of the pyrenoid, where it occupies a distinct radial subdomain. Its Rubisco-binding C-terminus is needed to concentrate it in the tubules. RBMP1 assembles in the membrane as pentamers, the typical bestrophin channel stoichiometry, and in larger complexes. It was first proposed to tether the Rubisco matrix to the tubules. However, mutants lacking the protein form normal pyrenoids with tubules and grow normally at air CO2, and the protein does not pull thylakoids into a reconstituted Rubisco condensate in Arabidopsis. Instead, the mutants have a transiently raised energy-dependent quenching during dark-to-light transitions, which points to a more acidic thylakoid lumen, and they grow poorly under fluctuating high light. The current view is therefore that RBMP1/BST4 is a pyrenoid tubule ion channel, possibly for bicarbonate, that helps to balance lumenal ion and proton levels during light changes. Its permeant ion has not been identified, since no currents were recorded in Xenopus oocytes.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005247 voltage-gated chloride channel activity | IBA GO_REF:0000033 | MODIFY | Summary: Phylogenetic transfer from Arabidopsis VCCN1 (AT3G61320). RBMP1/BST4 does have a conserved bestrophin channel domain and forms pentamers, so assigning channel activity to the family is reasonable. However, the chloride selectivity and voltage gating are not supported for this protein. BST4 has diverged at the selectivity pore, and no current was recorded in Xenopus oocytes with KCl or NaHCO3. Reason: The IBA places RBMP1 in the right channel family, and the bestrophin-domain evidence supports it. But the specific claim, a voltage-gated chloride channel, is the VCCN1 phenotype and was not reproduced for BST4, which has a pore substitution (Phe instead of the Val conserved in BST1-3). Its permeant ion is unknown, and the authors favour HCO3- as a hypothesis. Generalizing to monoatomic ion channel activity keeps the inherited channel function without asserting chloride selectivity or voltage gating. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: FUNCTIONAL DIVERGENCE GRANULARITY MISMATCH Sources checked: AGI_LocusCode:AT3G61320 Β· AtVCCN1 SUPPORTS SOURCE BUT NOT TARGET VCCN1 is an experimentally characterized voltage-dependent chloride channel. BST4 falls in a separate green-algal clade with a divergent pore residue, and no chloride current was detected. PANTHER:PTN004990962 Β· PTN004990962 SUPPORTS TRANSFER The channel activity is plausibly ancestral to the bestrophin/YneE node. Only the chloride and voltage-gating specificity is over-scoped for this member. Proposed replacements: monoatomic ion channel activity Supporting Evidence: PMID:39240724 BST4 also has a well-conserved bestrophin domain similar to those in the thylakoid-localized BST1-3 proteins PMID:39240724 Second, BST4 has a phenylalanine residue in the first position of the putative selection pore, as opposed to valine which is conserved throughout BST1-3 PMID:39240724 Therefore, we were unable to conclude what BST4 is permeable to. PMID:39240724 Based on these findings, we conclude that BST4 is a pentameric transmembrane channel found within the pyrenoid tubules but is not crucial for Rubisco matrix tethering. |
| GO:0005886 plasma membrane | IEA GO_REF:0000044 | REMOVE | Summary: The UniProt "Cell membrane" location comes from an ARBA rule for the YneE/bestrophin family, not from any observation of this protein. Every experimental localization places RBMP1/BST4 in thylakoid-derived pyrenoid tubule membranes inside the chloroplast. Reason: The rule-based plasma membrane location is contradicted by direct imaging in two independent studies, which find the protein in the pyrenoid tubules. In Arabidopsis, fractionation found it in thylakoid stroma lamellae and not in other membranes. Supporting Evidence: PMID:33177094 Fluorescently tagged RBMP1 and RBMP2 localized to the tubules PMID:39240724 The protein BST4 (bestrophin-like protein 4, also known as Rubisco binding membrane protein 1, RBMP1; Cre06.g261750) localizes exclusively to the pyrenoid tubules |
| GO:0019684 photosynthesis, light reaction | IBA GO_REF:0000033 | ACCEPT | Summary: Inherited from the VCCN1 clade, whose members modulate the thylakoid proton motive force during illumination. For RBMP1/BST4 there is matching target-specific evidence. bst4 mutants show transiently raised qE-type NPQ at dark-to-light transitions, consistent with altered lumenal pH, and grow poorly under fluctuating high light. Complementation with full-length BST4 rescues these phenotypes. Reason: As a thylakoid (pyrenoid tubule) ion channel that affects lumenal ion and proton balance during illumination, BST4 takes part in the light reactions in the same way as VCCN1. The IBA is consistent with the mutant physiology. A more specific term, such as acclimation to fluctuating light, is not yet justified by mechanism. Supporting Evidence: PMID:39240724 We found that under fluctuating high light, bst4 had a growth defect compared to the WT and the line complemented with full-length BST4 PMID:39240724 BST4 influences the ion homeostasis and subsequently the pH of the pyrenoid tubules through its function as an ion channel, particularly during fluctuating light. |
| GO:0042651 thylakoid membrane | IBA GO_REF:0000033 | ACCEPT | Summary: Correct at the general level. The pyrenoid tubules are continuous with, and derived from, the thylakoid membrane. When BST4 lacks its C-terminus it spreads throughout the thylakoid membrane, and in Arabidopsis it sits in thylakoid stroma lamellae. The specific location in Chlamydomonas is the pyrenoid tubule, which is added below as a NEW annotation. Reason: The phylogenetic inference agrees with direct localization of BST4 to thylakoid-derived tubule membranes, and to thylakoids when expressed heterologously. Supporting Evidence: PMID:39240724 BST4ΞC-term-mScarlet expressed in the bst4 mutant line did not localize to the pyrenoid tubules but was found throughout the thylakoid membrane PMID:39240724 indicating that BST4 is located in the tubules and not the Rubisco-enriched pyrenoid matrix |
| GO:1902476 chloride transmembrane transport | IEA GO_REF:0000108 | MARK AS OVER ANNOTATED | Summary: This annotation is inferred logically from the IBA voltage-gated chloride channel activity. Chloride transport by RBMP1/BST4 has not been shown. No current was detected with 100 mM KCl in oocytes, and the authors' hypothesis is HCO3- permeation. Reason: The parent MF is being generalized to monoatomic ion channel activity because chloride selectivity is unsupported. The derived chloride-transport process therefore over-commits to a substrate that has not been demonstrated. It is not definitively wrong, since bestrophins often pass Cl-, but it should not be presented as known. Supporting Evidence: PMID:39240724 Therefore, we were unable to conclude what BST4 is permeable to. |
| GO:0160223 pyrenoid tubule | IDA PMID:39240724 Bestrophin-like protein 4 is involved in photosynthetic accl... | NEW | Summary: Direct localization of fluorescently tagged and antibody-detected RBMP1/BST4 to the pyrenoid tubules, by three independent studies. Reason: This is the most specific and best-supported location of the protein, and it is not covered by any existing annotation. Fluorescent-protein fusions (PMID:33177094, PMID:39240724), dual tagging against the Rubisco matrix (PMID:39240724) and expansion immunomicroscopy (PMID:42465278) all agree. Supporting Evidence: PMID:39240724 indicating that BST4 is located in the tubules and not the Rubisco-enriched pyrenoid matrix PMID:33177094 Fluorescently tagged RBMP1 and RBMP2 localized to the tubules PMID:42465278 The distribution of the transmembrane BST438,39 occupied an intermediate radial distribution |
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Download this section (compressed HTML)Q: Is RBMP1/BST4 functionally redundant with RBMP2 or other RBM-containing tubule proteins as a matrix-tubule tether? Would an rbmp1 rbmp2 double mutant disturb the tubule-matrix interface even though the single bst4 mutant does not?
Suggested experts: Mackinder LCM, Jonikas MC
Q: What does BST4 conduct (HCO3-, Cl-, organic anions or cations), and what activates it, given that no oocyte currents were recorded even after C-terminal truncation?
Suggested experts: Walker CE, Burlacot A
Q: Should the CCM module record RBMP1 and BST4 as one gene product (Cre06.g261750) and change its role from "Rubisco-binding tether" to "RBM-targeted pyrenoid tubule bestrophin channel"?
Experiment: Reconstitute purified BST4 pentamers in planar lipid bilayers or liposomes (with flux assays for HCO3-, Cl- and organic anions) across a range of pH values, with and without Rubisco binding to the C-terminus. Test the Phe pore residue by mutating it to Val, the residue found in BST1-3.
Hypothesis: BST4 is a bicarbonate-permeable channel that is active only under specific lumenal pH or with a partner protein.
Type: electrophysiology / reconstitution
Experiment: Generate rbmp1 rbmp2 double mutants (and RBM-mutated versions) and image tubule-matrix contacts by cryo-ET and live imaging. Measure CCM performance at limiting CO2.
Hypothesis: RBMP1 and RBMP2 act redundantly to tether the Rubisco matrix to the tubules.
Type: genetics / imaging
Experiment: Use a lumen-targeted pH sensor, or measure CAH3-dependent CO2 release, in wild type, bst4 and complemented lines during light transitions and fluctuating light.
Hypothesis: BST4 activity changes lumenal pH in the tubules during dark-to-light transitions.
Type: physiology
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