Sphingolipid-Enriched Microdomains in the Plasma Membrane of Saccharomyces Cerevisiae: Ergosterol-Free «Lipid Rafts» in the Gel Phase

Abstract

The plasma membrane (PM) of Saccharomyces cerevisiae was studied using the probes trans-parinaric acid (t-PnA) and diphenylhexatriene (DPH), providing the first direct evidence for the presence of gel domains in living cells and showing that the PM is mostly constituted by ordered domains(1). The fluorescence lifetimes of t-PnA are particularly sensitive to the presence and nature of ordered domains, and here the first measurements in yeast cells are reported. A long fluorescence lifetime typical of the gel phase (> 30 ns) was found in mid-exponential phase wild-type (wt) cells from two different genetic backgrounds, at both 24 and 30°C. The confirmation of the gel nature was achieved by comparing t-PnA and DPH fluorescence anisotropy, and studying the thermotropic transitions of liposomes reconstituted from PM lipid-extracts. To address the location and composition of the domains spheroplasts, the isolated PM, liposomes from total lipid-extracts, and living cells of several mutant strains with deletions in genes coding for enzymes involved in ergosterol-, sphingolipid- and GPI-anchor biosynthesis were also studied. It was shown that the gel domains are not ergosterol-enriched lipid rafts, but mainly composed of sphingolipids and GPI-anchored proteins, suggesting important roles in membrane traffic, signaling, and interactions with the cell wall. The findings provide a biophysical mechanism for the targeting of some proteins to the PM, which is sphingolipid-dependent but sterol-independent and for the formation of membrane compartments in the yeast PM (MCP and MCC)(2). Acknowledgements: PTDC/QUI-BIQ/104311/2008, Ciência2007 and PEst-OE/QUI/UI0612/2011 (FCT, Portugal). (1) F. Aresta-Branco, A.M. Cordeiro, H.S. Marinho, L. Cyrne, F. Antunes, R.F.M. de Almeida (2011) J. Biol. Chem. 286:5043-5054. (2) Poolman, B. (2011) Evaluation of: [Aresta-Branco F et al. Gel domains in the plasma membrane ...]. Faculty of 1000, 26 Apr 2011. F1000.com/9922956.