Lipid Phase Behavior of the Curvature Region of Thylakoid Membranes of Spinacia oleracea. Isotropic Phase around the CURT1 Protein

Abstract

Thylakoid membranes (TMs) of oxygenic photosynthetic organisms are flat membrane vesicles, which form highly organized, interconnected membrane networks. In vascular plants, they are differentiated into stacked and unstacked regions, the grana and stroma lamellae, respectively; they are densely packed with protein complexes performing the light reactions of photosynthesis and generating a proton motive force (pmf). The maintenance of pmf and its utilizations for ATP synthesis require sealing the TMs at their highly curved regions (CRs). These regions are devoid of chlorophyll-containing proteins but contain the curvature inducing CURVATURE THYLAKOID1 (CURT1) proteins, and are enriched in lipids. Because of the highly curved nature of this region, at the margins of grana and stroma TMs, the molecular organization of lipid molecules is likely to possess distinct features compared to those in the major TM domains. To clarify this question, we isolated CR fractions from Spinacia oleracea and, using BN-PAGE and western blot analysis, verified that they are enriched in CURT1 proteins and in lipids. The lipid phase behavior of these fractions was fingerprinted with 31P-NMR spectroscopy, which revealed that the bulk lipid molecules assume a non-bilayer, isotropic lipid phase. This finding underpins the importance of the main, non-bilayer lipid species, monogalactosyldiacylglycerol, of TMs in their self-assembly and functional activity.