Lipid Phase Behaviour of the Curvature Region of Thylakoid Membranes of Spinacia oleracea

Abstract

Thylakoid membranes (TMs) of oxygenic photosynthetic organisms are flat membrane vesicles, which form highly organised, 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 utilisation for ATP synthesis requires 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 organisation 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 behaviour 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.

Keywords: 31P‐NMR; CURT1 protein; granum margin; non‐bilayer lipid phase; thylakoid membrane.

FIGURE 1

(A) lpBN‐PAGE analysis of TM and fractions after solubilization with 0.4% digitonin of spinach (So) TM. At: Arabidopsis thaliana . (B) immunoblot with PsaB, Cp47and CURT1A antibodies of the same TM and fractions as in (A). A cross‐reactivity scale with So TM is shown on the left.

FIGURE 2

SEM micrographs of CR fractions showing bent membrane particles (A) which often coagulate (B).

FIGURE 3

³¹P‐NMR spectra of CR under control conditions, displaying also the effects of saturation pulses (SP) at 2.7 ppm (A) and 0.15 ppm (B). The number of scans was 12,800.

FIGURE 4

³¹P‐NMR spectra of untreated (Control) and WGL‐treated (20 U mL⁻¹, 25,600 scans) (A) and trypsin‐treated (10 mg mL⁻¹, 19,200 scans) (B) CR samples.