Isolation and characterization of lipid rafts in E miliania huxleyi : a role for membrane microdomains in host–virus interactions

Abstract

Summary Coccolithoviruses employ a suite of glycosphingolipids ( GSLs ) to successfully infect the globally important coccolithophore E miliania huxleyi . Lipid rafts, chemically distinct membrane lipid microdomains that are enriched in GSLs and are involved in sensing extracellular stimuli and activating signalling cascades through protein–protein interactions, likely play a fundamental role in host–virus interactions. Using combined lipidomics, proteomics and bioinformatics, we isolated and characterized the lipid and protein content of lipid rafts from control E . huxleyi cells and those infected with EhV 86, the type strain for C occolithovirus . Lipid raft‐enriched fractions were isolated and purified as buoyant, detergent‐resistant membranes ( DRMs ) in OptiPrep density gradients. Transmission electron microscopy of vesicle morphology, polymerase chain reaction amplification of the EhV major capsid protein gene and immunoreactivity to flotillin antisera served as respective physical, molecular and biochemical markers. Subsequent lipid characterization of DRMs via high performance liquid chromatography‐triple quadrapole mass spectrometry revealed four distinct GSL classes. Parallel proteomic analysis confirmed flotillin as a major lipid raft protein, along with a variety of proteins affiliated with host defence, programmed cell death and innate immunity pathways. The detection of an EhV 86‐encoded C ‐type lectin‐containing protein confirmed that infection occurs at the interface between lipid rafts and cellular stress/death pathways via specific GSLs and raft‐associated proteins.

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