Final Report Summary - PROTEOLIPIDOMICS1 (Proteolipidomic characterization of the regulatory circuitry of global lipid metabolism) The lipidome of eukaryotic cells consists of hundreds to thousands of lipid species that constitute membranes, store metabolic energy and function as bioactive signaling molecules. The compositional diversity of lipids is produced by a metabolic network that interconnects the metabolism of fatty acids, glycerophospholipids, glycerolipids, sphingolipids, and sterol lipids. This network enables dynamic modulation of the lipidome to support remodeling of cellular processes and architecture in physiological adaptive processes. Dysfunctional regulation of lipid metabolism and homeostasis causes cellular lipotoxicity, impairs cellular processes and contributes to the pathogenesis of disorders such as obesity, atherosclerosis, and neurodegeneration. How lipid complexity affects cell physiology and how cells regulate lipid metabolism on a lipidome-wide level remain unclear.The main aim of this project was to develop and apply a mass spectrometry (MS)-based proteolipidomics strategy for systems-level characterization of the regulatory circuitry of lipid metabolism in the yeast Saccharomyces cerevisiae. To this end, we first developed a proteomics routine for the comprehensive and quantitative analysis of the yeast proteome. Specifically, we optimized yeast lysis and protein extraction, protein digestion, liquid chromatographic separation of peptides and MS parameters. The resulting analytical approach afforded the accurate quantification of ~3700 yeast proteins in a biological experiment.Having established the analytical strategy for quantification of the yeast proteome, we combined comprehensive quantitative proteomics and lipidomics to analyze how global lipid metabolism and cellular processes are dynamically co-regulated during physiological adaptations. For this, we performed a time series analysis of the S. cerevisiae growth profile covering conserved metabolic programs and physiological transitions. This experimental framework enabled the comparison between fermentative and respiratory metabolism as well as between proliferating and quiescent cells in a physiological context. In higher eukaryotes, the ability to alternate between quiescence and proliferation regulates development and tissue homeostasis whereas the switch between metabolic programs impinges on cell growth and is central to proliferative diseases such as cancer.Our results provided the first comprehensive reference of lipid and protein dynamics in a eukaryote and afforded unique insights that demonstrate coordination of global lipid metabolism with the functional restructuring of cellular architecture and processes. In particular, we demonstrate that the lipid enzymatic machinery is reprogrammed to activate cardiolipin synthesis and remodeling to support mitochondrial function during cellular respiration, that lipid droplet-associated triacylglycerols and sterol esters undergo distinct cycles of storage and mobilization, that sphingolipid composition is dynamically adjusted in a previously unidentified growth stage-specific manner, and that endogenous modulation of fatty acid unsaturation participates in the control of peroxisomal biogenesis in vivo. Importantly, defects in cardiolipin remodeling, peroxisome function or lipid droplet homeostasis have been associated to human pathologies including the Barth syndrome, obesity or neurological disorders. Besides, the lipid droplet-related triacylglycerol synthesis is prominently linked to oil production (e.g. for seed oils or biofuels). Hence, our results may potentially contribute to future biotechnology and health research.Besides revealing the functional plasticity of the lipid metabolic network, our work also provides a new experimental and analytical framework for investigating the physiological control of lipid metabolism at the global scale. This platform is generic and can in future be applied in yeast to investigate the regulation and function of conserved lipid enzymes and signaling pathways using genetic and pharmacological interventions or be translated to higher eukaryotes. Ongoing applications of the proteolipidomics platform will focus on regulation of global lipid metabolism during cell cycle progression.