Amphipol‐assisted purification method for the highly active and stable photosystem <scp>II</scp> supercomplex of <i>Chlamydomonas reinhardtii</i>

Watanabe, Akimasa; Kim, Eun Chul; Burton‐Smith, Raymond N.; Tokutsu, Ryutaro; Minagawa, Jun

doi:10.1002/1873-3468.13394

Cited by 27 publications

(38 citation statements)

References 41 publications

(59 reference statements)

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“…In this work, we used a combination of mild detergent (digitonin and α-DM) and APols to efficiently isolate and stabilise LHCII complexes in different native states (i.e. trimers and large aggregates) (40,41,44), aiming to isolate the LHCII aggregates within the thylakoid membrane during NPQ formation and thereby validating the aggregation model. The main advantage of using this polymers over detergents alone is being able to isolate large protein complexes in their close-to-native form.…”

Section: Discussionmentioning

confidence: 99%

“…The main advantage of using this polymers over detergents alone is being able to isolate large protein complexes in their close-to-native form. Previously, APols have been used to investigate structural and functional aspects of membrane proteins, including photosynthetic complexes (44)(45)(46)66). APols bind non-covalently to the hydrophobic surface of the membrane proteins with an extremely slow dissociation rate (40).…”

Section: Discussionmentioning

confidence: 99%

“…This makes their association with membrane proteins more stable (67-69), even at extreme dilution (69). Upon being trapped with APols, detergentsolubilized membrane proteins remain in the native conformational organisation that they were in before detergent removal (40,44,45,70). Therefore, the use of APols rules out the possibility of artificially-induced aggregation of the membrane proteins.…”

Section: Discussionmentioning

confidence: 99%

“…S2). We then used a combination of detergents to solubilize the thylakoid membranes, which were subsequently replaced by APols A8-35 (1% w/v) to stabilise and isolate LHCII complexes before dissociation (41,44) (Fig. S2).…”

Section: Isolation Of Lhcii Aggregates From Nom Thylakoid Membranes Lmentioning

confidence: 99%

“…While these polymers are not particularly efficient at solubilising biological membranes, they are capable to trap effectively solubilised membrane proteins and keep them stable in their native conformation (42,43). Ionic and non-ionic APols have been successfully used to stabilise highly active PSII-LHCII supercomplexes of Chlamydomonas reinhardtii in their major organisation (C2S2M2L2) (43), revealing high resolution structures of these supercomplexes (44)(45)(46).…”

Section: Introductionmentioning

confidence: 99%

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A novel method produces native light-harvesting complex II aggregates from the photosynthetic membrane revealing their role in nonphotochemical quenching

Shukla

Watanabe

Wilson

et al. 2020

Journal of Biological Chemistry

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Non-photochemical quenching (NPQ) is a mechanism of regulating light harvesting that protects the photosynthetic apparatus from photodamage by dissipating excess absorbed excitation energy as heat. In higher plants, the major light-harvesting antenna complex (LHCII) of photosystem (PS) II is directly involved in NPQ. The aggregation of LHCII is proposed to be involved in quenching. However, the lack of success in isolating native LHCII aggregates has limited the direct interrogation of this process. The isolation of LHCII in its native state from thylakoid membranes has been problematic due to the use of detergent, which tends to dissociate loosely-bound proteins, and the abundance of pigment-protein complexes (e.g. PSI and PSII) embedded in the photosynthetic membrane, which hinders the preparation of aggregated LHCII. Here, we used a novel purification method employing detergent and amphipols to entrap LHCII in its natural states. To enrich the photosynthetic membrane with the major LHCII, we used Arabidopsis thaliana plants lacking the PSII minor antenna complexes (NoM), treated with lincomycin to inhibit the synthesis of PSI and PSII core proteins. Using sucrose density gradients, we succeeded in isolating the trimeric as well as aggregated forms of LHCII antenna. Violaxanthin- and zeaxanthin-enriched complexes were investigated in dark-adapted, NPQ, and dark recovery states. Zeaxanthin-enriched antenna complexes showed the greatest amount of aggregated LHCII. Notably, the amount of aggregated LHCII decreased upon relaxation of NPQ. Employing this novel preparative method, we obtained a direct evidence for the role of in vivo LHCII aggregation in NPQ.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Isolation Of Lhcii Aggregates From Nom Thylakoid Membranes Lmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A novel method produces native light-harvesting complex II aggregates from the photosynthetic membrane revealing their role in nonphotochemical quenching

Shukla

Watanabe

Wilson

et al. 2020

Journal of Biological Chemistry

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Structural characterization of the photosystems in the green alga Chlorella sorokiniana

Watanabe

Minagawa

2020

Planta

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Formation of a PSI–PSII megacomplex containing LHCSR and PsbS in the moss Physcomitrella patens

et al. 2019

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Mosses are one of the earliest land plants that diverged from freshwater green algae. They are considered to have acquired a higher capacity for thermal energy dissipation to cope with dynamically changing solar irradiance by utilizing both the "algal-type" light-harvesting complex stress-related (LHCSR)-dependent and the "plant-type" PsbS-dependent mechanisms. It is hypothesized that the formation of photosystem (PS) I and II megacomplex is another mechanism to protect photosynthetic machinery from strong irradiance. Herein, we describe the analysis of the PSI-PSII megacomplex from the model moss, Physcomitrella patens, which was resolved using large-pore clear-native polyacrylamide gel electrophoresis (lpCN-PAGE). The similarity in the migration distance of the Physcomitrella PSI-PSII megacomplex to the Arabidopsis megacomplex shown during lpCN-PAGE suggested that the Physcomitrella PSI-PSII and Arabidopsis megacomplexes have similar structures. Time-resolved chlorophyll fluorescence measurements show that excitation energy was rapidly and efficiently transferred from PSII to PSI, providing evidence of an ordered association of the two photosystems. We also found that LHCSR and PsbS co-migrated with the Physcomitrella PSI-PSII megacomplex. The megacomplex showed pH-dependent chlorophyll fluorescence quenching, which may have been induced by LHCSR and/or PsbS proteins with the collaboration of zeaxanthin. We discuss the mechanism that regulates the energy distribution balance between two photosystems in Physcomitrella.

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Amphipol‐assisted purification method for the highly active and stable photosystem II supercomplex of Chlamydomonas reinhardtii

Cited by 27 publications

References 41 publications

A novel method produces native light-harvesting complex II aggregates from the photosynthetic membrane revealing their role in nonphotochemical quenching

A novel method produces native light-harvesting complex II aggregates from the photosynthetic membrane revealing their role in nonphotochemical quenching

Structural characterization of the photosystems in the green alga Chlorella sorokiniana

Formation of a PSI–PSII megacomplex containing LHCSR and PsbS in the moss Physcomitrella patens

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