Coupling between sterol and sphingolipid structure in ordered membrane domains

A hallmark of eukaryotic membranes is the pairing of lineage-specific sterols with characteristic sphingolipid species. Mammalian cell membranes are enriched in both cholesterol and long-chain sphingolipids such as sphingomyelin, whereas fungi synthesize ergosterol and very-long-chain sphingolipids with sugar-containing head groups. It has been proposed that these two lipid classes co-evolved to support membrane structure and organization. Here, we investigated how sterol structure and sphingolipid chain length together control membrane order and phase behavior. In the yeast Saccharomyces cerevisiae, loss of very-long-chain C26 sphingolipids disrupted formation of liquid-ordered (L_o) domains in the vacuole membrane. Similarly, replacing ergosterol synthesis with that of cholesterol also prevented vacuole L_o domains from forming. To determine a possible physical basis of these effects, we investigated synthetic membranes of defined composition containing either ergosterol or cholesterol and sphingomyelin with different chain lengths. In membranes containing egg sphingomyelin with C16 chains, ergosterol only sparsely supported L_o domains, in contrast to cholesterol. Membranes containing sphingomyelin with C26 chains displayed a different pattern. Cholesterol mixtures were largely homogeneous across most compositions, with only a limited region that supported fluid domains. Ergosterol mixtures exhibited a distinct compositional window that supported fluid domains positioned between regimes of uniform membranes and gel phases. This window corresponded to lipidome changes in the vacuole as it phase separates during nutritional restriction. Measurements of membrane order showed that cholesterol strongly increased membrane packing compared to ergosterol in membranes containing egg sphingomyelin, whereas this difference was lost in membranes containing C26 sphingomyelin. The results suggest that sphingolipid chain length can tune sterol interactions needed for membrane organization.