SAGA1 and SAGA2 localize the starch sheath to the pyrenoid in Chlamydomonas reinhardtii
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Abstract
Most algae enhance their CO 2 assimilation by concentrating CO 2 within the pyrenoid, a biomolecular condensate that contains the CO 2 -fixing enzyme Rubisco. Many pyrenoids are surrounded by a starch sheath that is thought to slow the escape of CO 2 from the pyrenoid, but how the starch sheath is localized to the pyrenoid remains poorly understood. Here, in the leading model alga Chlamydomonas reinhardtii , we find that the protein SAGA2 is necessary for early pyrenoid starch sheath biogenesis and works redundantly with its homolog, SAGA1, to localize the starch sheath to the pyrenoid. SAGA2 and SAGA1 were enriched in different regions of the pyrenoid-starch sheath interface, suggesting that they play complementary roles. Both saga2 and saga1 mutants showed defects in starch sheath coverage early during pyrenoid formation that were improved at a later timepoint. Strikingly, a saga1;saga2 double mutant did not have a starch sheath around the pyrenoid and showed decreased overall starch content. SAGA1 and SAGA2 starch-binding domains bound to starch, the starch mimic molecule β-cyclodextrin, and the starch precursor molecule maltoheptaose, suggesting a role for SAGA1 and SAGA2 in starch granule initiation. We propose a model where SAGA1 and SAGA2 each locally prime starch sheath initiation in a distinct region of the pyrenoid surface by enriching starch precursor molecules around the pyrenoid. These findings advance the understanding of algal starch sheath biogenesis and provide insights into the associations between biomolecular condensates and other cellular structures.
Significance Statement
Eukaryotic algae enhance their carbon assimilation using an organelle called the pyrenoid, where concentrated CO 2 is supplied to the CO 2 -fixing enzyme Rubisco. In many algae, a starch sheath surrounding the pyrenoid is thought to enhance CO 2 fixation, but how starch is localized to pyrenoids is unknown. Here, we show that two proteins, SAGA1 and SAGA2, each bind to starch precursor molecules and redundantly localize starch to the pyrenoid in the alga Chlamydomonas reinhardtii . Our results suggest that SAGA1 and SAGA2 promote starch sheath initiation at the pyrenoid, rather than merely tethering starch, as previously thought. This work advances the understanding of the proteins and molecular mechanisms involved in pyrenoid starch sheath biogenesis and lays the foundations for their further study.
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Together, these findings establish that SAGA1 and SAGA2 can each compensate for the loss of the other to localize starch sheaths to the pyrenoid, but no other factor can compensate for the loss of both SAGA1 and SAGA2.
Do you think the redundancy is quantitative (dosage) or qualitative (distinct biochemical roles) (do SAGA1 and SAGA2 differ in binding kinetics, substrate specificity, or temporal expression?). A follow up rescue experiment swapping domains or expression timing could distinguish these.
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Our results indicate that whereas SAGA1 is enriched at the starch-tubule-matrix junctions of the pyrenoid, SAGA2 is depleted from these regions, validating previous observations and supporting the idea that SAGA1 and SAGA2 play complementary roles in starch sheath biogenesis
What do you think defines these spatial domains? Are they driven by intrinsic protein differences (e.g., phase separation propensity, binding affinities) or by pre-existing pyrenoid substructure (e.g., Rubisco density gradients, thylakoid tubules)? Maybe quantitative co-localization with known pyrenoid subcompartments or perturbation of pyrenoid architecture could clarify causality. Correlative light em microscopy or spatial proteomics might help resolve whether these domains correspond to structural features (e.g., thylakoid proximity) or represent emergent …
Our results indicate that whereas SAGA1 is enriched at the starch-tubule-matrix junctions of the pyrenoid, SAGA2 is depleted from these regions, validating previous observations and supporting the idea that SAGA1 and SAGA2 play complementary roles in starch sheath biogenesis
What do you think defines these spatial domains? Are they driven by intrinsic protein differences (e.g., phase separation propensity, binding affinities) or by pre-existing pyrenoid substructure (e.g., Rubisco density gradients, thylakoid tubules)? Maybe quantitative co-localization with known pyrenoid subcompartments or perturbation of pyrenoid architecture could clarify causality. Correlative light em microscopy or spatial proteomics might help resolve whether these domains correspond to structural features (e.g., thylakoid proximity) or represent emergent patterning driven by SAGA proteins themselves (though i acknowledge these are way beyond the scope of this current study)
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