Lipid Polymorphism of the Subchloroplast—Granum and Stroma Thylakoid Membrane–Particles. II. Structure and Functions

Abstract

In Part I, by using 31P-NMR spectroscopy, we have shown that isolated granum and stroma thylakoid membranes (TMs), in addition to the bilayer, display two isotropic phases and an inverted hexagonal (HII) phase; saturation transfer experiments and selective effects of lipase and thermal treatments have shown that these phases arise from distinct, yet interconnectable structural entities. To obtain information on the functional roles and origin of the different lipid phases, here we performed spectroscopic measurements and inspected the ultrastructure of these TM fragments. Circular dichroism, 77 K fluorescence emission spectroscopy, and variable chlorophyll-a fluorescence measurements revealed only minor lipase- or thermally induced changes in the photosynthetic machinery. Electrochromic absorbance transients showed that the TM fragments were re-sealed, and the vesicles largely retained their impermeabilities after lipase treatments—in line with the low susceptibility of the bilayer against the same treatment, as reflected by our 31P-NMR spectroscopy. Signatures of HII-phase could not be discerned with small-angle X-ray scattering—but traces of HII structures, without long-range order, were found by freeze-fracture electron microscopy (FF-EM) and cryo-electron tomography (CET). EM and CET images also revealed the presence of small vesicles and fusion of membrane particles, which might account for one of the isotropic phases. Interaction of VDE (violaxanthin de-epoxidase, detected by Western blot technique in both membrane fragments) with TM lipids might account for the other isotropic phase. In general, non-bilayer lipids are proposed to play role in the self-assembly of the highly organized yet dynamic TM network in chloroplasts.

Structural Entities Associated with Different Lipid Phases of Plant Thylakoid Membranes—Selective Susceptibilities to Different Lipases and Proteases

“…The addition of 20 U mL −1 WGL further suppressed the isotropic phases and diminished the L and H II phases; at 100 U mL −1 of WGL, all phases were eliminated ( Figure 6 ). These data are in good agreement with our previous results on isolated granum and stroma sub-chloroplast TM particles [ 38 , 39 ]. It is important to note that 5 U mL −1 WGL, even over long incubation times, did not disturb the structure and function of TMs; it did not increase the permeability of the membranes and exerted no effects on the excitation-energy distribution in the TMs, on the activity of PSII, nor the pattern and macro-organization of PPCs ( Figure S4 ).…”

“…In our earlier studies, we established correlations among the variations in the temperature, pH, and osmotic and ionic strengths of the medium as well as the activity of VDE and the phase behavior of the bulk lipid molecules [ 31 , 36 , 37 ]; we also clarified that isolated grana and stroma lamellae contain all the three non-bilayer phases [ 38 ]. Analyses of our structural and functional data strongly suggested that the isotropic phases originate from membrane fusions and junctions [ 39 ] and from VDE: lipid assemblies [ 31 ]. (Note that in model membranes, VDE: lipid structures form an H II phase [ 40 ].)…”

Non-bilayer Lipid Phases Modulate the Structure and Functions of Mitochondrial Membranes: Pharmacological Relevance

“…On the other hand, the lipocalin-non-bilayer lipid structures may rejoin the bilayer membrane network of TM or IMM when the remodelling and extension of the membrane network occurs. Isolated granum and stroma TMs placed in a proper aqueous environment spontaneously form extended and interconnected membrane networks made of narrow membrane channels rich in proteins embedded in the bilayer membrane [30]. Thus, DEM proposes the coexistence of the bilayer and non-bilayer phases in the TM and IMM and serves to explain the dynamic homeostasis of the energy-transducing membranes in which the equilibrium may shift towards the bilayer or non-bilayer phase depending on the energy needs of cell [9].…”