Abstract
The yeast vacuole is the only in vivo system where micron-scale liquid ordered (Lo) domains or lipid rafts can be readily observed. Upon nutrient depletion in stationary phase, yeast actively tunes its vacuole membrane to form Lo domains that serve as docking sites for lipid droplet stores. However, it is unknown what specific lipid components drive phase separation. Sphingolipids have been implicated as key components for lipid raft formation in artificial membranes, so we investigated the role of sphingolipids in vacuole domain formation. Three glycosylated sphingolipids are biosynthesized in yeast: inositol phosphorylceramide (IPC), mannose inositol phosphorylceramide (MIPC) and mannose diinositol phosphorylceramide (M(IP)2C). Lipidomics analysis of biochemically isolated stationary phase vacuoles revealed a dramatic increase (>2-fold) in these complex sphingolipids compared to exponential phase vacuoles. This change was not present in whole cell lipidomes of the same cells, indicating that sphingolipids get sorted into the vacuole during stationary phase. To test the role of different sphingolipids in vacuole phase separation, we systematically engineered sphingolipid composition in a series of strains. Reduction of IPC levels through modulation of AUR1 expression strongly inhibited domain formation. In cells lacking the MIPC and M(IP)2C, vacuole domains showed altered morphologies compared to wild-type cells. Our results show that the metabolism and trafficking of complex sphingolipids is a key driver of membrane phase separation in yeast vacuole.
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