Revisiting the expression and purification of MGD1, the major galactolipid synthase in Arabidopsis to establish a novel standard for biochemical and structural studies

Rocha, Joana; Audry, Magali; Pesce, Gaelle; Chazalet, V.; Block, Maryse A.; Maréchal, Éric; Breton, C.

doi:10.1016/j.biochi.2012.11.011

Cited by 13 publications

(29 citation statements)

References 24 publications

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“…1 A and ref. 13). The monomeric state, with a Stockes radius of 3.12 nm, was confirmed by size‐exclusion chromatography (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…MGD1 sequence beginning at E137 and fused to a His 6 ‐tag at the C terminus was expressed in E. coli using the pET expression system (Novagen; Merck‐Millipore, Darmstadt, Germany) and purified by a 2‐step procedure, including immobilized metal affinity chromatography and size exclusion chromatography as detailed previously (13). MGD1 activity was checked in vitro as described previously (12).…”

Section: Methodsmentioning

confidence: 99%

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The influence of lipids on MGD1 membrane binding highlights novel mechanisms for galactolipid biosynthesis regulation in chloroplasts

et al. 2014

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Mono- and digalactosyldiacylglycerol (MGDG and DGDG) are the most abundant lipids of photosynthetic membranes (thylakoids). In Arabidopsis green tissues, MGD1 is the main enzyme synthesizing MGDG. This monotopic enzyme is embedded in the inner envelope membrane of chloroplasts. DGDG synthesis occurs in the outer envelope membrane. Although the suborganellar localization of MGD1 has been determined, it is still not known how the lipid/glycolipid composition influences its binding to the membrane. The existence of a topological relationship between MGD1 and "embryonic" thylakoids is also unknown. To investigate MGD1 membrane binding, we used a Langmuir membrane model allowing the tuning of both lipid composition and packing. Surprisingly, MGD1 presents a high affinity to MGDG, its product, which maintains the enzyme bound to the membrane. This positive feedback is consistent with the low level of diacylglycerol, the substrate of MGD1, in chloroplast membranes. By contrast, MGD1 is excluded from membranes highly enriched in, or made of, pure DGDG. DGDG therefore exerts a retrocontrol, which is effective on the overall synthesis of galactolipids. Previously identified activators, phosphatidic acid and phosphatidylglycerol, also play a role on MGD1 membrane binding via electrostatic interactions, compensating the exclusion triggered by DGDG. The opposite effects of MGDG and DGDG suggest a role of these lipids on the localization of MGD1 in specific domains. Consistently, MGDG induces the self-organization of MGD1 into elongated and reticulated nanostructures scaffolding the chloroplast membrane.-Sarkis, J., Rocha, J., Maniti, O., Jouhet, J., Vié, V., Block, M. A., Breton, C., Maréchal, E., Girard-Egrot, A. The influence of lipids on MGD1 membrane binding highlights novel mechanisms for galactolipid biosynthesis regulation in chloroplasts.

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“…1 A and ref. 13). The monomeric state, with a Stockes radius of 3.12 nm, was confirmed by size‐exclusion chromatography (Fig.…”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The influence of lipids on MGD1 membrane binding highlights novel mechanisms for galactolipid biosynthesis regulation in chloroplasts

et al. 2014

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“…The generation of the catalytic domain protein construct (cdMGD1) has been described elsewhere (Rocha et al ., ). Briefly, the catalytic domain of MGD1 (residues 137–533), with a C–terminal His 6 tag, was cloned into pET29b vector (Novagen, http://www.novagen.com/).…”

Section: Methodsmentioning

confidence: 97%

Structural insights and membrane binding properties of MGD1, the major galactolipid synthase in plants

et al. 2016

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). The structures of cdMGD1 and cdMGD1:UDP have been deposited in the Protein Data Bank under accession numbers 4WYI and 4X1T, respectively. SUMMARYMonogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the major lipid components of photosynthetic membranes, and hence the most abundant lipids in the biosphere. They are essential for assembly and function of the photosynthetic apparatus. In Arabidopsis, the first step of galactolipid synthesis is catalyzed by MGDG synthase 1 (MGD1), which transfers a galactosyl residue from UDP-galactose to diacylglycerol (DAG). MGD1 is a monotopic protein that is embedded in the inner envelope membrane of chloroplasts. Once produced, MGDG is transferred to the outer envelope membrane, where DGDG synthesis occurs, and to thylakoids. Here we present two crystal structures of MGD1: one unliganded and one complexed with UDP. MGD1 has a long and flexible region (approximately 50 amino acids) that is required for DAG binding. The structures reveal critical features of the MGD1 catalytic mechanism and its membrane binding mode, tested on biomimetic Langmuir monolayers, giving insights into chloroplast membrane biogenesis. The structural plasticity of MGD1, ensuring very rapid capture and utilization of DAG, and its interaction with anionic lipids, possibly driving the construction of lipoproteic clusters, are consistent with the role of this enzyme, not only in expansion of the inner envelope membrane, but also in supplying MGDG to the outer envelope and nascent thylakoid membranes.

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“…Biochemical data obtained on the purified spinach enzyme showed a sequential, random or ordered, mechanism with independent donor and acceptor binding sites ( Maréchal et al, 1994a ). Major advance was recently obtained in the production and crystallization of the catalytic domain of MGD1 ( Rocha et al, 2013 , 2016 ). The recombinant protein produced in Escherichia coli is fully active but it behaves as a monomer in solution and not as a dimer as previously proposed ( Miège et al, 1999 ).…”

Section: The Mgdg Synthasesmentioning

confidence: 99%

Do Galactolipid Synthases Play a Key Role in the Biogenesis of Chloroplast Membranes of Higher Plants?

et al. 2018

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A unique feature of chloroplasts is their high content of the galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), which constitute up to 80% of their lipids. These galactolipids are synthesized in the chloroplast envelope membrane through the concerted action of galactosyltransferases, the so-called ‘MGDG synthases (MGDs)’ and ‘DGDG synthases (DGDs),’ which use uridine diphosphate (UDP)-galactose as donor. In Arabidopsis leaves, under standard conditions, the enzymes MGD1 and DGD1 provide the bulk of galactolipids, necessary for the massive expansion of thylakoid membranes. Under phosphate limited conditions, plants activate another pathway involving MGD2/MGD3 and DGD2 to provide additional DGDG that is exported to extraplastidial membranes where they partly replace phospholipids, a phosphate-saving mechanism in plants. A third enzyme system, which relies on the UDP-Gal-independent GGGT (also called SFR2 for SENSITIVE TO FREEZING 2), can be activated in response to a freezing stress. The biosynthesis of galactolipids by these multiple enzyme sets must be tightly regulated to meet the cellular demand in response to changing environmental conditions. The cooperation between MGD and DGD enzymes with a possible substrate channeling from diacylglycerol to MGDG and DGDG is supported by biochemical and biophysical studies and mutant analyses reviewed herein. The fine-tuning of MGDG to DGDG ratio, which allows the reversible transition from the hexagonal II to lamellar α phase of the lipid bilayer, could be a key factor in thylakoid biogenesis.

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Revisiting the expression and purification of MGD1, the major galactolipid synthase in Arabidopsis to establish a novel standard for biochemical and structural studies

Cited by 13 publications

References 24 publications

The influence of lipids on MGD1 membrane binding highlights novel mechanisms for galactolipid biosynthesis regulation in chloroplasts

The influence of lipids on MGD1 membrane binding highlights novel mechanisms for galactolipid biosynthesis regulation in chloroplasts

Structural insights and membrane binding properties of MGD1, the major galactolipid synthase in plants

Do Galactolipid Synthases Play a Key Role in the Biogenesis of Chloroplast Membranes of Higher Plants?

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