Analysis of sphingolipid-signaling at the plasma membrane of Saccharomyces cerevisiae
The protein and lipid composition of eukaryotic plasma membranes is highly dynamic\nand regulated according to need. Despite its great plasticity, the plasma membrane\nretains some organizational features, such as its lateral organization into distinct\ndomains. In the yeast, Saccharomyces cerevisiae, large immobile protein clusters,\ntermed eisosomes, are important for plasma membrane organization. Eisosomes\nhelp to sort proteins into discrete domains, function in endocytosis and are implicated\nin cellular signaling. The major eisosome components Pil1 and Lsp1 were first\nidentified as in vitro targets of the sphingolipid long chain base-regulated Pkhkinases.\nHowever, it is not known if eisosomes are targets of Pkh-mediated\nsphingolipid signaling in vivo. In this thesis, I show that Pkh-kinases regulate\neisosome formation in response to alterations of complex sphingolipid levels in the\nplasma membrane. I found that Pkh-kinase-dependent phosphorylation of Pil1\ncontrols the assembly state of eisosomes. The combination of different unbiased,\nglobal analysis methods, such as proteomics and high content screening enabled me\nto identify Nce102 as a negative regulator of Pkh-kinases. Nce102 relocalizes\nbetween MCC domains, overlaying eisosomes, and the remainder of the plasma\nmembrane in response to alterations in sphingolipid levels. Together with its\nregulatory function on Pkh-kinases that localize at eisosomes, this relocalization\nsuggests that it is part of a sphingolipid sensor. Furthermore, I identified Rom2, a\nRho1 GTPase exchange factor, as a novel regulator of sphingolipid metabolism. My\ndata reveal several new insights into regulation of sphingolipid metabolism and\nplasma membrane organization. I provide a model how a homeostatic feedback loop\nmay control sphingolipid levels. This likely will help in understanding how cells adjust\nprocesses, such as eisosome driven domain organization, endocytosis and actin\norganization to altered conditions. Furthermore, I anticipate that the datasets created\nin this thesis will serve as a resource for future studies on plasma membrane\nfunction.