Functional convergence of hopanoids and sterols in membrane ordering

Abstract

Liquid-ordered phases are one of two biochemically active membrane states, which until now were thought to be a unique consequence of the interactions between eukaryotic membrane lipids. The formation of a liquid-ordered phase depends crucially on the ordering properties of sterols. However, it is not known whether this capacity exists in organisms that lack sterols, such as bacteria. We show that diplopterol, the simplest bacterial hopanoid, has similar properties and that hopanoids are bacterial "sterol surrogates" with the ability to order saturated lipids and to form a liquid-ordered phase in model membranes. These observations suggest that the evolution of an ordered biochemically active liquid membrane could have evolved before the oxygenation of Earth's surface and the emergence of sterols.

Fig. 1.

Structures of cholesterol and diplopterol (A) and a conceptual cartoon illustrating liquid-disordered (L_d), liquid-ordered (L_o), and gel phase membranes (B).

Fig. 2.

The effects of cholesterol and diplopterol on SM. (A) Monolayers of SM and mixtures containing cholesterol (chol) or diplopterol (dip) were compressed at 25 °C. The condensation effect of chol and dip on SM was calculated as shown to the right of the isotherm traces. (B) The membrane ordering effect (∆GP) of chol or dip on SM was determined by C-laurdan spectroscopy on liposomes labeled with 0.2 mol% C-laurdan and composed of SM, SM/chol (2:1 mol%), and SM/dip (2:1) at 50 °C. The ∆GP was calculated as the difference in GP between liposomes containing pure SM and mixtures of either SM/chol or SM/dip.

Fig. 3.

Diplopterol forms a liquid ordered phase. (A) Confocal images of GUVs at 22 °C and labeled with 0.2 mol% C-laurdan. The composition (mol %) of GUVs is given below each image, and GP values are depicted by color. (B) Average GP of one- and two-component GUVs and of ordered and disordered domains from three-component GUVs (n = 4). (C) Autocorrelation curves and estimated diffusion times of Atto532 labeled sphingomyelin (0.001 mol%) in SM/Chol, SM/Dip, and DOPC GUVs at 22 °C.

Fig. 4.

Ordering effect of diplopterol (dip) and cholesterol (chol) on kdo-lipid A. (A) comparison of the structures of kdo-lipid A and SM. (B) Isotherms of Lipid A mixtures and calculated condensation effect of chol and dip on kdo-lipid A at 25 °C and pH 7.4, 5.1, and 3.1. (C) The membrane order (GP) of liposomes labeled with 0.2 mol% C-laurdan containing kdo-lipid A, kdo-lipid A/dip, and kdo-lipid A/chol. The pH-induced change in relative membrane order was calculated as the difference in GP (GP_pH3 – GP_pH7) between pH 3 and 7 for the three mixtures at 25 °C.