Sec14, the major yeast phosphatidylinositol (PtdIns)/phosphatidylcholine (PtdCho) transfer protein, regulates essential interfaces between lipid metabolism and membrane trafficking from the trans-Golgi network (TGN). How Sec14 does so remains unclear. We report that Sec14 binds PtdIns and PtdCho at distinct (but overlapping) sites, and both PtdIns- and PtdCho-binding activities are essential Sec14 activities. We further show both activities must reside within the same molecule to reconstitute a functional Sec14 and for effective Sec14-mediated regulation of phosphoinositide homeostasis in vivo. This regulation is uncoupled from PtdIns-transfer activity and argues for an interfacial presentation mode for Sec14-mediated potentiation of PtdIns kinases. Such a regulatory role for Sec14 is a primary counter to action of the Kes1 sterol-binding protein that antagonizes PtdIns 4-OH kinase activity in vivo. Collectively, these findings outline functional mechanisms for the Sec14 superfamily and reveal additional layers of complexity for regulating phosphoinositide homeostasis in eukaryotes.

We report the lipidomic response of the murine macrophage RAW cell line to Kdo 2 -lipid A, the active component of an inflammatory lipopolysaccharide functioning as a selective TLR4 agonist and compactin, a statin inhibitor of cholesterol biosynthesis. Analyses of lipid molecular species by dynamic quantitative mass spectrometry and concomitant transcriptomic measurements define the lipidome and demonstrate immediate responses in fatty acid metabolism represented by increases in eicosanoid synthesis and delayed responses characterized by sphingolipid and sterol biosynthesis. Lipid remodeling of glycerolipids, glycerophospholipids, and prenols also take place, indicating that activation of the innate immune system by inflammatory mediators leads to alterations in a majority of mammalian lipid categories, including unanticipated effects of a statin drug. Our studies provide a systems-level view of lipid metabolism and reveal significant connections between lipid and cell signaling and biochemical pathways that contribute to innate immune responses and to pharmacological perturbations.The "omics" revolution has provided significant insight into the genes, mRNAs, and proteins of mammalian cells, biological systems, and disease (1-3). An important function of these macromolecular classes is the production of metabolites that in turn are used by cells for replication and function. Lipids comprise major structural and metabolic components of cells and have essential functions in the formation of membranes, energy production, and intracellular signaling. Despite the central role of lipids in mammalian cell function, there has been no systematic effort to define the lipid "parts list" of a mammalian cell or the changes in these lipids associated with cellular function and disease. Many biochemical pathways leading to the synthesis and degradation of major lipid categories are known, but how these pathways interact under normal and pathological conditions remains unexplored. Recent advances in mass spectrometry have made it possible to qualitatively and quantitatively analyze a majority of cellular lipids (4 -8). We report here a comprehensive systems-level analysis of a mammalian cell lipidome through temporal measurements.We characterized lipidomic responses of RAW264.7 (RAW) macrophages to a highly specific ligand for Toll-like receptor 4 (TLR4) 4 that mimics aspects of bacterial infection. This model is of particular interest because of the essential roles that alterations in macrophage lipid metabolism play in innate and adaptive immune responses and the development of chronic inflammatory and cardiovascular diseases. Recent studies further suggest that TLR signaling in macrophages is not only required for innate immunity against viral and bacterial pathogens but also contributes to the pathogenesis of atherosclerosis, diabetes, arthritis, and other inflammatory diseases (9). Although TLR4 signaling is known to exert profound effects on the macrophage transcriptome (10), proteome (11), and selected lipid species that...

The LIPID MAPS Consortium (www.lipidmaps. org) is developing comprehensive procedures for identifying all lipids of the macrophage, following activation by endotoxin. The goal is to quantify temporal and spatial changes in lipids that occur with cellular metabolism and to develop bioinformatic approaches that establish dynamic lipid networks. To achieve these aims, an endotoxin of the highest possible analytical specification is crucial. We now report a large-scale preparation of 3-deoxy-D-manno-octulosonic acid (Kdo) 2 -Lipid A, a nearly homogeneous Re lipopolysaccharide (LPS) sub-structure with endotoxin activity equal to LPS. Kdo 2 -Lipid A was extracted from 2 kg cell paste of a heptose-deficient Escherichia coli mutant. It was purified by chromatography on silica, DEAE-cellulose, and C18 reverse-phase resin. Structure and purity were evaluated by electrospray ionization/mass spectrometry, liquid chromatography/mass spectrometry and 1 H-NMR. Its bioactivity was compared with LPS in RAW 264.7 cells and bone marrow macrophages from wild-type and toll-like receptor 4 (TLR-4)-deficient mice. Cytokine and eicosanoid production, in conjunction with gene expression profiling, were employed as readouts. Kdo 2 -Lipid A is comparable to LPS by these criteria. Its activity is reduced by . The LIPID MAPS consortium is developing quantitative methods for evaluating the composition, biosynthesis, and function of all macrophage lipids (1). These amphipathic substances not only are structural components of biological membranes but also play important roles in the pathophysiology of inflammation, atherosclerosis, and growth control. Additional lipid functions should emerge from the comprehensive analysis of macrophage lipids. Electrospray ionization/mass spectrometry (ESI/MS) (2, 3), coupled with prefractionation methods like reversephase liquid chromatography (LC), is being applied systematically to set the stage for the seamless integration of lipid metabolism into the broader fields of genomics, proteomics, and systems biology. To facilitate this endeavor, LIPID MAPS has introduced a new comprehensive classification system for biological lipids, amenable to computer-based data processing and substructure comparison (4). The eight LIPID MAPS categories are 1) fatty acyls, 2) glycerolipids, 3) glycerophospholipids, 4) sphingolipids, 5) sterol lipids, 6) prenol lipids, 7) saccharolipids,