rbcL is the chloroplast-genome-encoded large subunit (about 55 kDa, 475 residues) of form I ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) in Chlamydomonas reinhardtii. Eight large subunits, arranged as four head-to-tail homodimers, form the catalytic core of the L8S8 holoenzyme and are capped at each end by four nuclear-encoded small subunits. Each large-subunit dimer contributes the active sites, which bind Mg2+ and are activated by carbamylation of Lys-201. The enzyme catalyses the carboxylation of D-ribulose 1,5-bisphosphate to two molecules of 3-phosphoglycerate, the CO2-fixing step of the Calvin-Benson-Bassham (reductive pentose-phosphate) cycle, and, in competition at the same active site, the oxygenation of ribulose 1,5-bisphosphate that starts photorespiration. Crystal structures of the activated Chlamydomonas enzyme with the transition-state analogue 2-CABP show the conserved form I architecture and post-translational modifications including 4-hydroxyproline and S-methylcysteine. In the chloroplast the holoenzyme is distributed between the stroma and the pyrenoid, a liquid-like, non-membranous Rubisco condensate in which most of the enzyme is concentrated when cells grow at limiting CO2; recruitment to the pyrenoid is determined by the small subunit, which carries the binding sites for the linker protein EPYC1, not by the large subunit. Because rbcL is encoded in the chloroplast and is accessible by chloroplast transformation, Chlamydomonas rbcL has been a central system for directed mutagenesis of Rubisco catalysis and CO2/O2 specificity.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000287 magnesium ion binding | IEA GO_REF:0000120 | ACCEPT | Summary: Each Rubisco large subunit binds one Mg2+ in the active site, where it coordinates the carbamylated Lys-201 and the bound substrate. Mg2+ is seen directly in the crystal structures of the Chlamydomonas holoenzyme complexed with 2-CABP (UniProt cofactor annotation, evidence from PMID:11641402 and PMID:11866526). Reason: The InterPro/UniRule-based electronic annotation is correct for this protein. Mg2+ is an obligate catalytic cofactor of the large-subunit active site and is resolved in the Chlamydomonas crystal structures. The term records cofactor binding, which is part of how the carboxylase activity is carried out, rather than a separate function, so it is accepted without being listed as a separate core function. Supporting Evidence: PMID:11866526 The crystal structure of the complex of activated rubisco from the green alga Chlamydomonas reinhardtii and the reaction intermediate analogue 2-carboxyarabinitol-1,5-bisphosphate (2-CABP) has been solved at 1.84 A resolution file:CHLRE/rbcL/rbcL-uniprot.txt Note=Binds 1 Mg(2+) ion per subunit. |
| GO:0009507 chloroplast | IEA GO_REF:0000120 | ACCEPT | Summary: rbcL is encoded and translated in the chloroplast, and the holoenzyme is found in the chloroplast stroma and in the pyrenoid. Reason: Correct. Chloroplast is a general term, but it is accurate for an enzyme that is made and acts in the plastid. The more precise locations are the pyrenoid (added as NEW below) and the chloroplast stroma. The fraction in each depends on CO2 availability, about 90% in the pyrenoid at ambient CO2 and about 40% at elevated CO2 (PMID:9536077), so the general term is still a reasonable summary. Supporting Evidence: PMID:18664299 eight large subunits (~55 kDa, coded by the chloroplast rbcL gene) and eight small subunits (~15 kDa, coded by a family of nuclear rbcS genes) PMID:9536077 Using immunolocalization, the amount of Rubisco localized to the pyrenoid or to the chloroplast stroma was estimated for C. reinhardtii cells grown under different conditions. |
| GO:0015977 carbon fixation | IEA GO_REF:0000120 | ACCEPT | Summary: Rubisco performs the carboxylation step by which inorganic CO2 is incorporated into organic carbon, and the large subunit carries the active site that catalyses it. Reason: Correct, if general. The large subunit catalyses the CO2-fixing reaction itself, so it does the work of carbon fixation. The more specific process, GO:0019253 reductive pentose-phosphate cycle, is also annotated and is the one used in core_functions. Broad IEA terms are acceptable alongside the specific one. Supporting Evidence: PMID:11866526 Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) catalyzes the initial steps of photosynthetic carbon reduction and photorespiratory carbon oxidation cycles by combining CO(2) and O(2), respectively, with ribulose-1,5-bisphosphate. |
| GO:0016984 ribulose-bisphosphate carboxylase activity | IEA GO_REF:0000120 | ACCEPT | Summary: The large subunit carries the Rubisco active site, which sits at the interface of a head-to-tail large-subunit dimer and contains the carbamylated Lys-201 and Mg2+. Chlamydomonas Rubisco has been purified and assayed directly for carboxylase kinetics many times, including large-subunit mutants made by chloroplast transformation of an rbcL deletion strain. Reason: This is the defining catalytic activity of rbcL. The structural and kinetic evidence for Chlamydomonas Rubisco is extensive (PMID:11866526, PMID:18664299). The small subunit modulates catalysis but does not carry the active site (PMID:20424165), so the activity is correctly assigned to the large subunit with an "enables" relationship. The oxygenase reaction at the same site has no separate GO term (GO:0016984 lists "ribulose bisphosphate carboxylase/oxygenase activity" as an exact synonym). Supporting Evidence: PMID:20424165 Although the large subunit contains the active site, a family of rbcS nuclear genes encodes the Rubisco small subunits, which can also influence the carboxylation catalytic efficiency and CO(2)/O(2) specificity of the enzyme. PMID:18664299 The kinetic constants of purified and activated enzymes were determined by measuring the incorporation of acid-stable 14C from NaH14CO3 PMID:18664299 Mutant MX3312, which has the rbcL coding region replaced with the aadA gene conferring spectinomycin resistance, was used as the host for chloroplast transformation |
| GO:0019253 reductive pentose-phosphate cycle | IEA GO_REF:0000104 | ACCEPT | Summary: Rubisco catalyses the carboxylation step of the Calvin-Benson-Bassham cycle, converting ribulose 1,5-bisphosphate and CO2 into 3-phosphoglycerate. Reason: The large subunit catalyses the entry reaction of the cycle, so it takes part in the process directly, not just as a requirement. Rubisco mutants of Chlamydomonas cannot grow photosynthetically and must be kept on acetate, which agrees with this role. Supporting Evidence: PMID:11866526 Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) catalyzes the initial steps of photosynthetic carbon reduction and photorespiratory carbon oxidation cycles PMID:18664299 Mutants that lack Rubisco function can be maintained with acetate |
| GO:0009573 chloroplast ribulose bisphosphate carboxylase complex | IDA PMID:11641402 First crystal structure of Rubisco from a green alga, Chlamy... | NEW | Summary: Crystal structures of Rubisco purified from Chlamydomonas show the L8S8 holoenzyme, in which eight rbcL large subunits form the catalytic core and eight nuclear-encoded small subunits cap the ends. Reason: GOA has no complex annotation for P00877, although membership of the chloroplast L8S8 holoenzyme is shown directly by two independent crystal structures of the Chlamydomonas enzyme (PMID:11641402, PMID:11866526). UniProt records the subunit structure as a heterohexadecamer of 8 large and 8 small chains. GO:0009573 is the chloroplast-specific Rubisco complex term and is the correct one for a chloroplast-encoded large subunit. Supporting Evidence: PMID:11641402 The crystal structure of Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase) from the unicellular green alga Chlamydomonas reinhardtii has been determined to 1.4 A resolution. Overall, the structure shows high similarity to the previously determined structures of L8S8 Rubisco enzymes. PMID:11866526 Many photosynthetic organisms have form I rubiscos comprised of eight large (L) and eight small (S) subunits. PMID:11866526 The subunit arrangement of Chlamydomonas rubisco is the same as those of the previously solved form I rubiscos. |
| GO:1990732 pyrenoid | IDA PMID:9536077 The intracellular localization of ribulose-1,5-bisphosphate ... | NEW | Summary: Immunolocalization showed that most Chlamydomonas Rubisco is in the pyrenoid, about 90% in cells grown at ambient CO2 and about 40% at elevated CO2, with the rest in the stroma. Pyrenoidal Rubisco is catalytically active. Cryo-ET and live imaging show the pyrenoid matrix to be a liquid-like condensate of Rubisco holoenzymes and the linker EPYC1. Reason: Rubisco is the main constituent of the pyrenoid matrix, and it is the holoenzyme, which contains the large subunit, that is condensed there. Immunogold localization with anti-Rubisco antibodies places it in the pyrenoid (PMID:9536077), and condensed Rubisco remains functional in vitro (PMID:30498228). The large subunit does not cause the pyrenoid localization. Targeting to the pyrenoid is set by two surface alpha-helices of the small subunit (PMID:23112177, PMID:20424165), and EPYC1 binds the small subunit (PMID:33230314). This annotation therefore records where the large subunit carries out carboxylation, not a targeting or condensate-scaffolding role for rbcL. Some of the enzyme stays in the stroma, especially at high CO2. Supporting Evidence: PMID:9536077 It was observed that the amount of Rubisco in the pyrenoid varied with growth condition; about 40% was in the pyrenoid when the cells were grown under elevated CO2 and about 90% with ambient CO2. PMID:9536077 In addition, it is likely that pyrenoidal Rubisco is active in CO2 fixation because in vitro activity measurements showed that most of the Rubisco must be active to account for CO2-fixation rates observed in whole cells. PMID:30498228 The phase-separated Rubisco is functional. PMID:20424165 It appears that small subunits contain the structural elements responsible for targeting Rubisco to the algal pyrenoid |
| GO:0009570 chloroplast stroma | IDA PMID:9536077 The intracellular localization of ribulose-1,5-bisphosphate ... | NEW | Summary: Quantitative immunolocalization shows that the part of Rubisco not in the pyrenoid is in the chloroplast stroma. This is about 60% of the enzyme in cells grown at elevated CO2 and about 10% at ambient CO2. Reason: The stromal pool is a substantial and probably active share of Rubisco, especially when the CCM is not induced. Together with the pyrenoid annotation, this term describes where the large subunit acts more precisely than the general chloroplast IEA. It is a specific child of the accepted GO:0009507 chloroplast annotation, not a redundant one. Supporting Evidence: PMID:9536077 Using immunolocalization, the amount of Rubisco localized to the pyrenoid or to the chloroplast stroma was estimated for C. reinhardtii cells grown under different conditions. PMID:9536077 about 40% was in the pyrenoid when the cells were grown under elevated CO2 and about 90% with ambient CO2. |
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Download this section (compressed HTML)Q: Rubisco oxygenation of ribulose 1,5-bisphosphate is the first step of photorespiration and is catalysed by the same large-subunit active site. GO has no separate oxygenase activity term (the oxygenase is an exact synonym of GO:0016984). Should the large subunit carry GO:0009853 photorespiration, and how do Arabidopsis and other plant RBCL annotations handle this?
Q: Does the large subunit make any contribution to Rubisco-EPYC1 binding? Cryo-EM suggests a possible contact between EPYC1 R56 and large-subunit E433. Or is pyrenoid packing set entirely by the small-subunit alpha-helices?
Q: Is the stromal (non-pyrenoid) pool of Rubisco, which is larger at high CO2, catalytically different from the pyrenoid pool, or does only the CO2 supply differ?
Experiment: Express plant-like or carboxysome-linker-binding large-subunit variants by chloroplast transformation of the rbcL deletion strain MX3312, and measure pyrenoid formation and EPYC1 co-condensation. This would test directly whether the large-subunit surface affects pyrenoid partitioning.
Type: chloroplast transformation and imaging
Experiment: Compare in situ Rubisco activation state and kinetics in isolated pyrenoids and in stroma fractions from low- and high-CO2-grown cells.
Type: subcellular fractionation and enzyme kinetics
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