SAGA1 (StArch Granules Abnormal 1; locus Cre11.g467712) is a large (~1,626 aa, ~180 kDa) nuclear-encoded pyrenoid protein of Chlamydomonas reinhardtii. It is built from an N-terminal CBM20 starch-binding domain, a long coiled-coil/alpha-helical middle region, and disordered segments that carry two copies of the pyrenoid Rubisco-binding motif, one at the extreme C terminus. SAGA1 binds the Rubisco large and small subunits, and its Rubisco-binding motif peptides bind Rubisco in vitro. Its CBM20 domain binds starch granules, beta-cyclodextrin and maltoheptaose in vitro. In the cell SAGA1 sits at the pyrenoid periphery. It forms puncta and rings where the thylakoid membranes pass through gaps in the starch sheath and become the membrane tubules that traverse the Rubisco matrix, and it is also present along those matrix-traversing membranes. SAGA1 is needed to start these tubules. It binds its homologue MITH1 and recruits it, and MITH1 then extends the membranes through the matrix. Expressed together with MITH1 in an Arabidopsis line carrying an EPYC1-Rubisco condensate, SAGA1 is sufficient to make thylakoid membranes traverse the condensate. Its proposed role is to provide an adhesive bridge between the membrane and the Rubisco matrix. Working redundantly with its paralogue SAGA2, SAGA1 also places the starch sheath around the pyrenoid. The saga1 single mutant has abnormally elongated, thin starch plates. The saga1 saga2 double mutant lacks a pyrenoid starch sheath, and its starch is spread through the stroma. Mutants lacking SAGA1 form many (about ten) tubule-less pyrenoid condensates per chloroplast and have a defective CO2-concentrating mechanism. They grow poorly at limiting CO2 and also fail to express several CAS-dependent inorganic-carbon transporter genes.
Definition: The formation of pyrenoid tubules, the thylakoid membranes that enter and traverse the Rubisco matrix of a pyrenoid.
Justification: SAGA1 and MITH1 are necessary for pyrenoid tubules and together sufficient to make thylakoids traverse a Rubisco condensate in Arabidopsis. GO has a pyrenoid tubule cellular component term but no process term for its formation, so the role can only be captured with the generic thylakoid membrane organization term.
Parent term: thylakoid membrane organization
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0030246 carbohydrate binding | IEA GO_REF:0000002 | ACCEPT | Summary: SAGA1 has a CBM20 domain, and the purified domain binds starch granules, beta-cyclodextrin and maltoheptaose. Carbohydrate binding is correct but general. Reason: The InterPro carbohydrate-binding-like fold mapping is correct and is now supported by direct binding data (PMID:42090253). The more informative child term, GO:2001070 starch binding, is also annotated, so this broad IEA is harmless and can stay. Supporting Evidence: PMID:42090253 We conclude that both the SAGA1 CBM20 domain and the SAGA2 CBM20 domain can bind to not only starch but also to starch precursor molecules |
| GO:2001070 starch binding | IEA GO_REF:0000002 | ACCEPT | Summary: The CBM20 (IPR002044) mapping to starch binding is correct for SAGA1. All conserved CBM20 binding residues are present, and the purified SAGA1 CBM20 binds starch granules in vitro. Reason: In 2019 this rested on the domain prediction (PMID:31455733). It has since been confirmed experimentally with a tandem SAGA1 CBM20 construct, which bound waxy maize starch while the mVenus control did not, and whose binding was competed by beta-cyclodextrin and maltoheptaose (PMID:42090253). That direct assay (IDA-level evidence) used an isolated, duplicated domain rather than full-length SAGA1, which is normal for domain-level binding evidence. The in vivo phenotype agrees with it, since saga1 saga2 double mutants lose the pyrenoid starch sheath. Starch binding is a core function. It is how SAGA1 and SAGA2 hold the starch sheath at the pyrenoid. Supporting Evidence: PMID:31455733 All 5 highly conserved residues necessary for starch binding are present in SAGA1, suggesting that SAGA1 can bind starch PMID:42090253 We observed that both the 10×His-mVenus-2×SAGA1-CBM20 and the 10×His-mVenus-SAGA2-2×CBM20 proteins, but not a 10×His-mVenus control protein, bound to starch PMID:42090253 suggesting that SAGA1 and SAGA2 function redundantly to localize starch sheaths to the pyrenoid |
| GO:0160223 pyrenoid tubule | IDA PMID:39548241 SAGA1 and MITH1 produce matrix-traversing membranes in the C... | NEW | Summary: SAGA1-Venus localizes along the pyrenoid matrix-traversing membranes and is enriched in puncta and rings at their periphery, where thylakoid sheets enter the matrix and become tubules. It co-fractionates with membranes. Reason: Fluorescence co-localization with tubule chlorophyll, CAH3 and MITH1, plus membrane co-pelleting (PMID:39548241), show where SAGA1 acts in tubule initiation. Expansion microscopy (PMID:42465278) and Crans et al. (PMID:42090253) confirm enrichment at tubule entry sites. GO:0160223 is part_of GO:1990732 pyrenoid, so no separate NEW pyrenoid row is added. The pyrenoid-level location for the matrix-starch interface role is recorded in core_functions. Supporting Evidence: PMID:39548241 Our results indicate that in Chlamydomonas, SAGA1 localizes throughout the matrix-traversing membranes and is enriched in puncta at their periphery. PMID:42465278 The highly peripheral ring-like localization of SAGA1 at the entry sites of the cylindrical tubules suggests |
| GO:0010027 thylakoid membrane organization | IMP PMID:39548241 SAGA1 and MITH1 produce matrix-traversing membranes in the C... | NEW | Summary: SAGA1 is required to start pyrenoid tubules, the thylakoid membranes that traverse the Rubisco matrix. With MITH1 it is sufficient to make thylakoid membranes traverse an EPYC1-Rubisco condensate in Arabidopsis. Reason: GO has no term for pyrenoid tubule biogenesis. Thylakoid membrane organization is the closest existing process, because the tubules are thylakoid-derived membranes. Participation test. SAGA1 is not a substrate or an upstream requirement. It binds Rubisco through its Rubisco-binding motifs (PMID:31455733, PMID:33177094), associates with the membranes and binds MITH1. Its proposed role is to supply the membrane-matrix adhesive bond that draws thylakoids into the matrix, a structural contribution to the reorganisation step. Heterologous sufficiency (with MITH1) makes this more than necessity evidence. Comparator check. Arabidopsis proteins that physically shape thylakoids, such as VIPP1 and FZL, carry GO:0010027 in QuickGO. The part that is still uncharacterised is how SAGA1 contacts the membrane. Supporting Evidence: PMID:39548241 SAGA1 is necessary for the initiation of matrix-traversing membranes as recognized by CYN7 (Fig. 3m–o) and CAH3 (Extended Data Fig. 8). PMID:39548241 These results demonstrate that SAGA1 and MITH1 together are sufficient to generate matrix-traversing thylakoid membranes in a heterologous system. PMID:39548241 We propose that SAGA1 and MITH1 together provide such Rubisco-membrane bonds, making it energetically favourable for membranes to traverse the matrix (Fig. 3u). |
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Download this section (compressed HTML)Q: Should GO add a "pyrenoid tubule assembly" process term (for example is_a thylakoid membrane organization, or under pyrenoid organization) so that tubule biogenesis factors such as SAGA1, MITH1 and RBMP2 can be annotated more specifically than GO:0010027?
Suggested experts: Jonikas MC
Q: How does SAGA1 contact the membrane? It has no predicted transmembrane segment. Does it bind lipids directly, a thylakoid membrane protein, or only MITH1? The answer decides whether protein-membrane adaptor activity is the right molecular function.
Q: Does SAGA1 actively initiate starch granules at the pyrenoid surface (GO:0062052 starch granule initiation), as its maltoheptaose binding suggests? Or does it only capture and anchor granules made by other enzymes?
Q: Does full-length SAGA1, with its single CBM20, bind starch with useful affinity in vivo? The in vitro assay needed a tandem 2xCBM20 construct to raise sensitivity.
Experiment: Reconstitute purified SAGA1 (or fragments) with liposomes of thylakoid lipid composition and Rubisco, with or without MITH1. Measure membrane binding by flotation and Rubisco recruitment to vesicles. Test RBM point mutants for loss of tubule initiation in the saga1 background.
Hypothesis: SAGA1 bridges Rubisco and thylakoid membranes directly.
Type: biochemical reconstitution and complementation
Experiment: Complement saga1 and saga1;saga2 with SAGA1 carrying inactivating mutations of the conserved CBM20 aromatic residues. Score starch sheath coverage, tubule formation, pyrenoid number and growth at low CO2.
Hypothesis: Starch binding by the SAGA1 CBM20 is needed for starch sheath placement but not for tubule initiation.
Type: genetic complementation and electron microscopy
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: The membrane-binding mechanism of SAGA1 is unknown. It co-pellets with membranes and localizes to matrix-traversing membranes, but it has no transmembrane segment and no identified lipid- or membrane-protein-binding region.
OPEN BIOLOGY
What is known: SAGA1 binds Rubisco and MITH1 and is necessary to initiate pyrenoid tubules.
Provenance (the field's own admissions):
Gap: GO has no process term specific to pyrenoid tubule biogenesis, so SAGA1's role can only be captured as thylakoid membrane organization.
OPEN ONTOLOGY
What is known: SAGA1 and MITH1 are necessary and together sufficient for membranes to traverse the pyrenoid matrix.
What would resolve it: Request a new GO BP term "pyrenoid tubule assembly".
Provenance (the field's own admissions):
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