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

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

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

International audienceA 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

Mechanism of activation of plant monogalactosyldiacylglycerol synthase 1 (MGD1) by phosphatidylglycerol

International audienceMono- and digalactosyldiacylglycerol are essential galactolipids for the biogenesis of plastids and functioning of the photosynthetic machinery. In Arabidopsis, the first step of galactolipid synthesis is catalyzed by monogalactosyldiacylglycerol synthase 1 (MGD1), a monotopic protein located in the inner envelope membrane of chloroplasts, which transfers a galactose residue from UDP-galactose to diacylglycerol (DAG). MGD1 needs anionic lipids such as phosphatidylglycerol (PG) to be active, but the mechanism by which PG activates MGD1 is still unknown. Recent studies shed light on the catalytic mechanism of MGD1 and on the possible PG binding site. Particularly, Pro189 was identified as a potential residue implicated in PG binding and His155 as the putative catalytic residue. In the present study, using a multifaceted approach (Langmuir membrane models, atomic force microscopy, molecular dynamics; MD), we investigated the membrane binding properties of native MGD1 and mutants (P189A and H115A). We demonstrated that both residues are involved in PG binding, thus suggesting the existence of a PG-His catalytic dyad that should facilitate deprotonation of the nucleophile hydroxyl group of DAG acceptor. Interestingly, MD simulations showed that MGD1 induces a reorganization of lipids by attracting DAG molecules to create an optimal platform for binding

Influence of lipid packing on the membrane-binding properties of mycolactone.

<p>Change in surface pressure (Δπ, mN/m) when mycolactone interacts with mixed monolayers at different initial surface pressures (π_i, mN/m). The nature of the lipid membrane was as follows: (A) and (B) mixture 1 consisting of 39% POPC, 33% SM, 9% POPE, 19% Chol. (C) and (D) mixture 2 consisting of 48% POPC, 41% SM, 11% POPE (given in mol%). Experiments were performed at 20°C (A) and (C), or 25°C (B) and (D). Each point corresponds to an independent measurement with a new lipid monolayer formed on PBS subphase (pH 7.4). The final concentration of mycolactone was 60 nM. Representative data from two or three independent assays are shown.</p

Interaction of mycolactone with mixed monolayers in the presence or absence of cholesterol.

<p>Adsorption kinetics (π-<i>t</i>) curves of mycolactone on monolayers composed of (A) mixture 1 (39% POPC, 33% SM, 9% POPE, 19% Chol) or (B) mixture 2 (48% POPC, 41% SM, 11% POPE) at 20°C (solid line) or 25°C (dashed line). Mycolactone was injected (4.45 μL) into the PBS subphase (pH 7.4) beneath the monolayer compressed at an initial surface pressure π_i of 30 mN/m after a relaxation time of one hour (arrows). Surface area was kept constant during the run. The final concentration of mycolactone was 60 nM. Each measurement was performed at least three times for each condition, and a representative curve is presented here.</p

Influence of mycolactone interaction on the distribution of the liquid-ordered (L_o) phase in the membrane.

<p>Fluorescence images of monolayers consisting of 39% POPC, 33% SM, 9% POPE, 19% Chol and including 0.5% BODIPY-cholesterol (TopFluor Cholesterol), after the injection (4.45 μL) of ethanol (row a) or mycolactone (row b) into the PBS subphase (pH 7.4) beneath the interfacial film compressed at an initial surface pressure of 30 mN/m at 20°C. The injection was performed after a relaxation time of one hour, and surface area was kept constant (mobile barriers were stopped). The final concentration of mycolactone was 60 nM. Scale bar: 50 μm.</p

Detergent effect on mixed monolayers in the presence of cholesterol.

<p>Adsorption kinetics (π-t) curves of detergent on monolayers consisting of 39% POPC, 33% SM, 9% POPE, 19% Chol (mixture 1) at 20°C, and the corresponding BAM images. (A) Tween 20 or Triton X-100 was injected into the PBS subphase (pH 7.4) beneath the monolayer at a final concentration of 60 nM. (B) Tween 20 or Triton X-100 was injected into the PBS subphase (pH 7.4) beneath the monolayer at a constant final “Active concentration/CMC ratio” of 0.06. (C) BAM images corresponding to the adsorption kinetics (π-t) curves after the injection of Tween 20 (b) and (d), or Triton X-100 (c) and (e), at a constant final concentration of 60 nM (b) and (c), or an “Active concentration/CMC ratio” of 0.06 (d) and (e). (a) Images recorded after the injection of a 60nM mycolactone (final concentration). In all experiments, the monolayer was compressed at an initial surface pressure π_i of 30 mN/m and detergents were injected after a relaxation time of one hour (arrows). Surface area was kept constant during the run. Each measurement was performed at least three times for each condition, and a representative curve is presented here. Image scale: 483 × 383 μm².</p

Surface pressure (π)–molecular area (<i>A</i>) isotherms and corresponding BAM images of monolayers with and without cholesterol.

<p>(A) π-<i>A</i> isotherms of mixture 1 (39% POPC, 33% SM, 9% POPE, 19% Chol given in mol%) recorded at 20°C (solid line) or 25°C (dashed line). (B) π-<i>A</i> isotherms of mixture 2 (48% POPC, 41% SM, 11% POPE given in mol%) recorded at 20°C (dashed-dotted line) or 25°C (dotted line). (C) Comparison of isotherms of the above-mentioned monolayers. Isotherms were recorded on PBS subphase (pH 7.4). Each isotherm corresponds to the mean of three experiments. BAM images were recorded during compression of the monolayer at a constant rate of 0.045 nm².molecule^-1.min^-1. Images A and B were recorded at 20°C. The images obtained for C were identical for 20 and 25°C. The estimated error for monolayers is ±0.05 mN/m for π and ≤ 0.01 nm² for (A). Image scale: 483 × 383 μm².</p

PLoS Pathog

Mycolactone is a lipid-like endotoxin synthesized by an environmental human pathogen, Mycobacterium ulcerans, the causal agent of Buruli ulcer disease. Mycolactone has pleiotropic effects on fundamental cellular processes (cell adhesion, cell death and inflammation). Various cellular targets of mycolactone have been identified and a literature survey revealed that most of these targets are membrane receptors residing in ordered plasma membrane nanodomains, within which their functionalities can be modulated. We investigated the capacity of mycolactone to interact with membranes, to evaluate its effects on membrane lipid organization following its diffusion across the cell membrane. We used Langmuir monolayers as a cell membrane model. Experiments were carried out with a lipid composition chosen to be as similar as possible to that of the plasma membrane. Mycolactone, which has surfactant properties, with an apparent saturation concentration of 1 mu M, interacted with the membrane at very low concentrations (60 nM). The interaction of mycolactone with the membrane was mediated by the presence of cholesterol and, like detergents, mycolactone reshaped the membrane. In its monomeric form, this toxin modifies lipid segregation in the monolayer, strongly affecting the formation of ordered microdomains. These findings suggest that mycolactone disturbs lipid organization in the biological membranes it crosses, with potential effects on cell functions and signaling pathways. Microdomain remodeling may therefore underlie molecular events, accounting for the ability of mycolactone to attack multiple targets and providing new insight into a single unifying mechanism underlying the pleiotropic effects of this molecule. This membrane remodeling may act in synergy with the other known effects of mycolactone on its intracellular targets, potentiating these effects