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Fröhlich, Florian (2010): Analysis of sphingolipid-signaling at the plasma membrane of Saccharomyces cerevisiae. Dissertation, LMU München: Faculty of Biology



The protein and lipid composition of eukaryotic plasma membranes is highly dynamic and regulated according to need. Despite its great plasticity, the plasma membrane retains some organizational features, such as its lateral organization into distinct domains. In the yeast, Saccharomyces cerevisiae, large immobile protein clusters, termed eisosomes, are important for plasma membrane organization. Eisosomes help to sort proteins into discrete domains, function in endocytosis and are implicated in cellular signaling. The major eisosome components Pil1 and Lsp1 were first identified as in vitro targets of the sphingolipid long chain base-regulated Pkhkinases. However, it is not known if eisosomes are targets of Pkh-mediated sphingolipid signaling in vivo. In this thesis, I show that Pkh-kinases regulate eisosome formation in response to alterations of complex sphingolipid levels in the plasma membrane. I found that Pkh-kinase-dependent phosphorylation of Pil1 controls the assembly state of eisosomes. The combination of different unbiased, global analysis methods, such as proteomics and high content screening enabled me to identify Nce102 as a negative regulator of Pkh-kinases. Nce102 relocalizes between MCC domains, overlaying eisosomes, and the remainder of the plasma membrane in response to alterations in sphingolipid levels. Together with its regulatory function on Pkh-kinases that localize at eisosomes, this relocalization suggests that it is part of a sphingolipid sensor. Furthermore, I identified Rom2, a Rho1 GTPase exchange factor, as a novel regulator of sphingolipid metabolism. My data reveal several new insights into regulation of sphingolipid metabolism and plasma membrane organization. I provide a model how a homeostatic feedback loop may control sphingolipid levels. This likely will help in understanding how cells adjust processes, such as eisosome driven domain organization, endocytosis and actin organization to altered conditions. Furthermore, I anticipate that the datasets created in this thesis will serve as a resource for future studies on plasma membrane function.