Context
limiting CO2 (air-level or lower inorganic carbon)
Pyrenoid-based CO2-concentrating mechanismBiological Processpyrenoid_ccm
A biophysical CO2-concentrating mechanism in which bicarbonate is converted to CO2 inside a phase-separated Rubisco condensate, raising the CO2 concentration at Rubisco and suppressing its oxygenase reaction.
pyrenoid-based CO2-concentrating mechanism
pyrenoidGO:1990732
Context
limiting CO2 (air-level or lower inorganic carbon)
Connections
Stromal bicarbonate imported by HLA3 and LCIA is the substrate moved into the lumen.
Bicarbonate formed by LCIB/LCIC from leaked or incoming CO2 joins the stromal pool that enters the thylakoid lumen.
Lumenal bicarbonate is the substrate for CAH3.
CO2 released in the tubules diffuses into the Rubisco matrix.
Tubules that traverse the matrix place CAH3-generated CO2 at the Rubisco condensate, and RBMP2 builds the reticulated region where CAH3 concentrates (CAH3 is mislocalized in rbmp2); without tubules the matrix forms aberrant condensates and growth at limiting CO2 is impaired.
Slowing CO2 escape from the matrix raises the CO2 concentration at Rubisco.
CIA5 is required for low-CO2 induction of BST1-3.
Part 1: Rubisco condensation into the pyrenoid matrix
Rubisco condensate (pyrenoid matrix)Cellular Componentrubisco_condensate
Rubisco and the intrinsically disordered linker EPYC1 are necessary and sufficient to phase separate into a liquid-like matrix. EPYC1 repeats bind the two surface alpha-helices of the Rubisco small subunit.
PMID:27166422A repeat protein links Rubisco to form the eukaryotic carbon-concentrating organelle.
Identifies EPYC1 as a Rubisco-binding repeat protein required for normal pyrenoid formation and CCM function.
PMID:28938114The Eukaryotic CO(2)-Concentrating Organelle Is Liquid-like and Exhibits Dynamic Reorganization.
Shows that the pyrenoid matrix behaves as a liquid-like phase.
PMID:30498228The phase separation underlying the pyrenoid-based microalgal Rubisco supercharger.
Rubisco and EPYC1 are necessary and sufficient to phase separate in vitro, and the condensed Rubisco is functional.
PMID:23112177Rubisco small-subunit α-helices control pyrenoid formation in Chlamydomonas.
Two surface-exposed small-subunit alpha-helices determine pyrenoid formation; plant-like helices abolish the pyrenoid and the CCM.
PMID:33230314The structural basis of Rubisco phase separation in the pyrenoid.
Structural basis of EPYC1 binding to the Rubisco small subunit.
PMID:33298923Condensation of Rubisco into a proto-pyrenoid in higher plant chloroplasts.
EPYC1 plus a plant-algal hybrid Rubisco forms a single liquid-like condensate in Arabidopsis chloroplasts, completing engineering step 1 in a plant.
Annotons
Rubisco carboxylation in the pyrenoid matrix
rubisco_carboxylation
Participant: Protein Complex: chloroplast Rubisco holoenzyme (L8S8)
Protein Complex:
chloroplast Rubisco holoenzyme (L8S8)GO:0009573
Active units:
Rubisco large subunit
Participant: Gene Product: Chlamydomonas rbcL
Required Function:
ribulose-bisphosphate carboxylase activityGO:0016984
Role: Carries the carboxylase active site.
Rubisco small subunit
Participant: Gene Product: Chlamydomonas RBCS1
Role: Presents the two surface alpha-helices bound by the EPYC1 linker; these helices determine whether a pyrenoid forms.
Function
ribulose-bisphosphate carboxylase activityGO:0016984
Substrates:
CO2
D-ribulose 1,5-bisphosphate
Products:
3-phospho-D-glycerate
Processes
Locations
The CO2-fixing enzyme whose condensation into the matrix is what the CCM concentrates CO2 around.
EPYC1 multivalent Rubisco linker
epyc1_linker
Participant: Gene Product: Chlamydomonas EPYC1 (also known as LCI5)
Function
Locations
Intrinsically disordered protein with repeated Rubisco-binding regions that cross-links Rubisco holoenzymes to drive phase separation of the matrix.
Part 2: Thylakoid tubule association with the condensate
Pyrenoid tubule biogenesisCellular Componentpyrenoid_tubule_biogenesis
Thylakoid membranes enter the matrix as pyrenoid tubules, built in three stages. SAGA1 initiates tubules where thylakoids meet the matrix; its homolog MITH1, recruited by SAGA1, extends them through the matrix; and RBMP2 converts the central tubules into a narrow reticulated region that hosts CAH3. Proteins carrying the Rubisco-binding motif (RBM) were proposed to hold the tubules and matrix together, but neither tubule RBM membrane protein tested so far behaves as a tether: BST4 (also called RBMP1) is not needed for tubule-matrix contact, and RBMP2 still builds the reticulated region without its RBMs. SAGA1 remains the best candidate for the membrane-matrix bond.
PMID:33177094Assembly of the algal CO(2)-fixing organelle, the pyrenoid, is guided by a Rubisco-binding motif.
A shared Rubisco-binding motif targets proteins to the pyrenoid and is found on tubule proteins (RBMP1, RBMP2) and on matrix-starch interface proteins (SAGA1, SAGA2).
PMID:39211136Biogenesis, engineering and function of membranes in the CO (2) -fixing pyrenoid.
SAGA1 and MITH1 mediate pyrenoid tubule biogenesis and are sufficient to reconstitute pyrenoid-traversing membranes in Arabidopsis; tubule-deficient mutants grow poorly at limiting CO2.
PMID:39548241SAGA1 and MITH1 produce matrix-traversing membranes in the CO(2)-fixing pyrenoid.
Peer-reviewed version of the SAGA1/MITH1 tubule biogenesis work; SAGA1-Venus lines the matrix-traversing membranes, and in mith1 mutants membranes stop at the matrix surface.
PMID:42427617RBMP2 shapes specialized membranes for CO(2) delivery in the pyrenoid condensate.
Preprint. rbmp2 mutants lack the central reticulated tubule region, mislocalize CAH3 and grow poorly at very low CO2; the transmembrane and membrane-contact regions are required, the RBMs are not.
PMID:39240724Bestrophin-like protein 4 is involved in photosynthetic acclimation to light fluctuations in Chlamydomonas.
BST4 (RBMP1) is a pentameric bestrophin-like channel in the pyrenoid tubules; bst4 mutants still form tubules in contact with the matrix, so BST4 is not required for matrix tethering.
Annotons
BST4/RBMP1 tubule-resident bestrophin-family channel
bst4_tubule_channel
Participant: Gene Product: Chlamydomonas BST4 / RBMP1 (Cre06.g261750)
Function
monoatomic ion channel activityGO:0005216 Bestrophin-family pentameric channel; the permeant ion is unknown (no chloride or bicarbonate currents in Xenopus oocytes). Its stroma-facing RBMs bind the Rubisco small subunit and target it to the tubules. Locations
Marks the pyrenoid tubule membrane and is proposed to help balance lumen ion or pH homeostasis during light changes. It was a candidate tubule-matrix tether, but that role was not supported.
SAGA1 tubule initiation
saga1_tubule_initiation
Participant: Gene Product: Chlamydomonas SAGA1 (Cre11.g467712)
Function
protein-membrane adaptor activityGO:0043495 Tentative. SAGA1 binds Rubisco through its RBMs and co-pellets with membranes, and is proposed to supply the membrane-matrix bond; how it attaches to membranes is unknown (it has no transmembrane segment). Processes
Locations
Localizes where thylakoids transition into tubules and is necessary to initiate tubule formation; with MITH1 it is sufficient to draw thylakoids into a Rubisco condensate in Arabidopsis.
MITH1 tubule extension
mith1_tubule_extension
Participant: Gene Product: Chlamydomonas MITH1 / SAGA3 (Cre06.g259100)
Function
SAGA1-dependent membrane-matrix adhesion (no GO term asserted) Molecular activity not established. MITH1 co-pellets with membranes and binds SAGA1, which recruits it; it has no transmembrane segment or Rubisco-binding motif, and direct Rubisco binding is unconfirmed (negative yeast two-hybrid). It is proposed to raise SAGA1's membrane-matrix adhesion.
Processes
Locations
Extends the membranes through the matrix after SAGA1 initiates them; in mith1 mutants the membranes stop at the matrix surface.
RBMP2 reticulated tubule region biogenesis
rbmp2_reticulated_region_biogenesis
Participant: Gene Product: Chlamydomonas RBMP2 (Cre09.g416850)
Function
membrane-shaping protein (no GO term asserted) Transmembrane protein with a non-catalytic rhodanese domain (no cysteine; aspartate at the catalytic position). Membrane bending is proposed but untested, so GO:0180020 is not asserted.
Processes
Locations
Converts the central tubules into the narrow reticulated region that concentrates CAH3 for CO2 release into the matrix.
Part 3: Lumenal carbonic anhydrase in the pyrenoid tubules
Lumenal bicarbonate dehydration (CAH3)Reactionlumenal_co2_release
In the acidic thylakoid lumen of the tubules, CAH3 dehydrates bicarbonate to CO2, which diffuses into the surrounding Rubisco matrix. Under limiting CO2, CAH3 is phosphorylated and activated, and its share in the pyrenoid tubules is reported to rise (from about 19% to 37% of the total); a 2026 preprint instead finds it concentrated in the central tubules at both high and low CO2.
PMID:9482718A novel alpha-type carbonic anhydrase associated with the thylakoid membrane in Chlamydomonas reinhardtii is required for growth at ambient CO2.
CAH3 is a lumenal alpha-type carbonic anhydrase; complementation of the cia-3 mutant with cah3 restores growth at air CO2.
PMID:23139834Phosphorylation controls the localization and activation of the lumenal carbonic anhydrase in Chlamydomonas reinhardtii.
After transfer to low CO2, CAH3 is phosphorylated and this correlates with partial relocalization to the pyrenoid and a 5-6 fold activity increase.
Annotons
CAH3 lumenal carbonic anhydrase
cah3_lumenal_ca
Participant: Gene Product: Chlamydomonas CAH3
Function
carbonate dehydratase activityGO:0004089
Substrates:
bicarbonate
Products:
CO2
water
Locations
Converts lumenal bicarbonate to CO2 inside the tubules so that CO2 is released directly into the Rubisco matrix.
Part 4: Bicarbonate channels in the thylakoid membrane
Thylakoid bicarbonate entry (bestrophin-like channels)Transport Stepthylakoid_bicarbonate_entry
Three bestrophin-like paralogs (BST1-3), induced at low CO2 under CIA5 control and located on the thylakoid membrane, are proposed to move stromal bicarbonate into the lumen. No transport has been measured for BST1-3: the evidence is localization, CIA5-dependent induction, knockdown physiology and homology to bestrophin anion channels. GO:0160133 bicarbonate channel activity would be the precise term once conductance is shown.
PMID:31391312Thylakoid localized bestrophin-like proteins are essential for the CO(2) concentrating mechanism of Chlamydomonas reinhardtii.
BST1-3 localize to the thylakoid membrane, are CIA5-dependent and low-CO2-induced, and their knockdown impairs Ci uptake and growth at limiting CO2.
Annotons
Bestrophin-like thylakoid bicarbonate channel
bst_thylakoid_channel
Participant: Family: Chlamydomonas thylakoid bestrophin-like proteins BST1-3
Family:
Chlamydomonas thylakoid bestrophin-like proteins BST1-3
Function
monoatomic anion channel activityGO:0005253 Family-level inference from the bestrophin channel fold. Bicarbonate is the proposed permeant anion but has not been tested. Locations
Supplies the lumenal bicarbonate that CAH3 dehydrates.
Part 5: Starch sheath diffusion barrier
Starch sheath around the matrixCellular Componentstarch_sheath_barrier
Plates of starch around the matrix are proposed to slow CO2 leakage. SAGA1 and its homolog SAGA2, each with starch-binding CBM20 domains and Rubisco-binding motifs, link the matrix to the sheath; the saga1;saga2 double mutant has no starch sheath at all. SAGA1 also controls sheath morphology and pyrenoid number. Modeling predicts that some diffusion barrier is needed for an efficient PCCM. Whether starch itself is the main barrier is not settled.
PMID:31455733A Rubisco-binding protein is required for normal pyrenoid number and starch sheath morphology in Chlamydomonas reinhardtii.
saga1 mutants have multiple pyrenoids with abnormal starch sheaths and are impaired in CCM function.
PMID:42090253SAGA1 and SAGA2 localize the starch sheath to the pyrenoid in Chlamydomonas reinhardtii.
The SAGA1 CBM20 domain binds starch granules in vitro, supporting SAGA1 and SAGA2 as the links that place the starch sheath around the matrix.
PMID:35596080Modelling the pyrenoid-based CO(2)-concentrating mechanism provides insights into its operating principles and a roadmap for its engineering into crops.
Model predicts that a barrier to CO2 diffusion out of the matrix is needed for effective and energetically efficient operation.
Annotons
SAGA1 matrix-starch sheath linker
saga1_matrix_starch_linker
Participant: Gene Product: Chlamydomonas SAGA1 (Cre11.g467712)
Function
starch bindingGO:2001070 A purified tandem SAGA1 CBM20 construct binds starch granules, competed by beta-cyclodextrin and maltoheptaose (PMID:42090253); binding by the full-length protein has not been tested. Locations
Bridges the Rubisco matrix to the starch sheath through Rubisco-binding motifs and a CBM20 domain.
SAGA2 matrix-starch sheath linker
saga2_matrix_starch_linker
Participant: Gene Product: Chlamydomonas SAGA2 (Cre09.g394621)
Function
starch bindingGO:2001070 Purified SAGA2 with both native CBM20 domains binds starch granules, competed by beta-cyclodextrin and maltoheptaose. Locations
Covers the matrix surface between tubule entry sites and tethers starch to the matrix, redundantly with SAGA1.
Part 6: Stromal CO2 recapture by carbonic anhydrase
LCIB/LCIC CO2 recapture at the pyrenoid peripheryReactionstromal_co2_recapture
The LCIB/LCIC complex, members of a distinct beta-carbonic anhydrase group, gathers around the pyrenoid under limiting CO2. Purified Chlamydomonas LCIB, LCIC and the LCIB/LCIC complex were inactive in vitro; CA activity of LCIB rests on rescue of CA-deficient yeast and Arabidopsis mutants and on its beta-CA zinc site. It is proposed to hydrate CO2 leaking from the matrix, and CO2 entering the chloroplast, to bicarbonate that is then trapped in the stroma.
PMID:20660228Light and low-CO2-dependent LCIB-LCIC complex localization in the chloroplast supports the carbon-concentrating mechanism in Chlamydomonas reinhardtii.
LCIB interacts with LCIC and the complex co-localizes in the vicinity of the pyrenoid under low CO2 in the light.
PMID:27911826Structural insights into the LCIB protein family reveals a new group of β-carbonic anhydrases.
Crystal structures of Chlamydomonas LCIB and LCIC show beta-CA-like active sites. The CA-active homologs were from a diatom and a bacterium; the purified Chlamydomonas proteins were inactive.
PMID:24307449LCIB in the Chlamydomonas CO2-concentrating mechanism.
Review of genetic evidence that LCIB is required for CCM function.
Annotons
LCIB/LCIC stromal carbonic anhydrase complex
lcib_lcic_ca
Participant: Protein Complex: LCIB/LCIC complex
Protein Complex:
LCIB/LCIC complex
Active units:
LCIB
Participant: Gene Product: Chlamydomonas LCIB
Role: Beta-CA subunit required for CCM function; carries the carbonic anhydrase activity shown by heterologous rescue.
LCIC
Participant: Gene Product: Chlamydomonas LCIC
Role: LCIB paralog that forms the hetero-oligomeric complex and binds zinc; no CA activity has been shown for LCIC.
Function
carbonate dehydratase activityGO:0004089 In this step the physiological direction is CO2 hydration to bicarbonate. CA activity of the Chlamydomonas complex itself has not been robustly measured in vitro.
Substrates:
CO2
water
Products:
bicarbonate
Locations
Traps CO2 that escapes the matrix as bicarbonate, returning it to the thylakoid route.
Part 7: Bicarbonate uptake into the chloroplast (upstream supply) (optional)
Plasma membrane and chloroplast envelope bicarbonate uptakeTransport Stepci_uptake
At very low CO2, HLA3 at the plasma membrane and LCIA at the chloroplast envelope raise bicarbonate supply to the stroma. This is part of the full algal CCM but outside the minimal PCCM proposed for plants, where stromal bicarbonate is supplied by native stromal CA.
PMID:19321421Knockdown of limiting-CO2-induced gene HLA3 decreases HCO3- transport and photosynthetic Ci affinity in Chlamydomonas reinhardtii.
HLA3 knockdown combined with LCIA or LCIB loss sharply reduces Ci uptake and photosynthetic Ci affinity at very low CO2.
PMID:26538195Introducing an algal carbon-concentrating mechanism into higher plants: location and incorporation of key components.
Expression and localization of algal CCM components, including Ci transporters, in Nicotiana and Arabidopsis.
Annotons
HLA3 plasma membrane bicarbonate uptake
hla3_plasma_membrane_uptake
Participant: Gene Product: Chlamydomonas HLA3 (Cre02.g097800)
Function
bicarbonate transmembrane transporter activityGO:0015106 Proposed from knockdown and Ci-uptake phenotypes. Locations
Moves external bicarbonate into the cytosol.
LCIA chloroplast envelope bicarbonate uptake
lcia_envelope_uptake
Participant: Gene Product: Chlamydomonas LCIA (NAR1.2)
Function
bicarbonate transmembrane transporter activityGO:0015106 Locations
Moves cytosolic bicarbonate into the chloroplast stroma.
Part 8: Low-CO2 induction of CCM genes (regulation) (optional)
CIA5/CCM1-dependent low-CO2 inductionRegulatory Stepccm_induction
CIA5 (also called CCM1), a zinc-finger nuclear regulator, is needed to induce CCM genes, including the BST channels, and to form a normal pyrenoid at limiting CO2.
PMID:11287669Ccm1, a regulatory gene controlling the induction of a carbon-concentrating mechanism in Chlamydomonas reinhardtii by sensing CO2 availability.
Ccm1 restores active carbon transport, pyrenoid development, and CCM gene induction in the C16 mutant.
Annotons
CIA5/CCM1 low-CO2 regulator
cia5_regulator
Participant: Gene Product: Chlamydomonas CIA5/CCM1
Function
putative transcriptional regulator (no GO term asserted) Zinc-finger protein with a glutamine-rich region typical of transcriptional activators. Direct DNA binding has not been established, so no DNA-binding transcription factor activity term is asserted.
Processes
Turns on the CCM program, including pyrenoid components, when inorganic carbon is limiting.