The Nonbilayer Lipid MGDG and the Major Light-Harvesting Complex (LHCII) Promote Membrane Stacking in Supported Lipid Bilayers.

The thylakoid membrane of algae and land plants is characterized by its intricate architecture, comprising tightly appressed membrane stacks termed grana. The contributions of individual components to grana stack formation are not yet fully elucidated. As an in vitro model, we use supported lipid bilayers made of thylakoid lipid mixtures to study the effect of major light-harvesting complex (LHCII), different lipids, and ions on membrane stacking, seen as elevated structures forming on top of the planar membrane surface in the presence of LHCII protein. These structures were examined by confocal laser scanning microscopy, atomic force microscopy, and fluorescence recovery after photobleaching, revealing multilamellar LHCII-membrane stacks composed of connected lipid bilayers. Both native-like and non-native interactions between the LHCII complexes may contribute to membrane appression in the supported bilayers. However, applying in vivo-like salt conditions to uncharged glycolipid membranes drastically increased the level of stack formation due to enforced LHCII-LHCII interactions, which is in line with recent crystallographic and cryo-electron microscopic data [Wan, T., et al. (2014) Mol. Plant 7, 916-919; Albanese, P., et al. (2017) Sci. Rep. 7, 10067-10083]. Furthermore, we observed the nonbilayer lipid MGDG to strongly promote membrane stacking, pointing to the long-term proposed function of MGDG in stabilizing the inner membrane leaflet of highly curved margins in the periphery of each grana disc because of its negative intrinsic curvature [Murphy, D. J. (1982) FEBS Lett. 150, 19-26].

The non-bilayer lipid MGDG stabilizes the major light-harvesting complex (LHCII) against unfolding.

In the photosynthetic apparatus of plants a high proportion of LHCII protein is needed to integrate 50% non-bilayer lipid MGDG into the lamellar thylakoid membrane, but whether and how the stability of the protein is also affected is not known. Here we use single-molecule force spectroscopy to map the stability of LHCII against mechanical unfolding along the polypeptide chain as a function of oligomerization state and lipid composition. Comparing unfolding forces between monomeric and trimeric LHCII demonstrates that the stability does not increase significantly upon trimerization but can mainly be correlated with specific contact sites between adjacent monomers. In contrast, unfolding of trimeric complexes in membranes composed of different thylakoid lipids reveals that the non-bilayer lipid MGDG substantially increases the mechanical stability of LHCII in many segments of the protein compared to other lipids such as DGDG or POPG. We attribute these findings to steric matching of conically formed MGDG and the hourglass shape of trimeric LHCII, thereby extending the role of non-bilayer lipids to the structural stabilization of membrane proteins in addition to the modulation of their folding, conformation and function.