Acyltransferases in Yeast: regulation, interaction and physiological function
Work in our lab is focused on fatty acid and lipid metabolism in the yeast, Saccharomyces cerevisiae . Specifically, we are interested in the metabolic flux of fatty acids from their site of endogenous synthesis, or from external sources, and subsequent incorporation into cellular lipids. These reactions are catalyzed by various acyl-transferases, which play an important role in defining the specific acyl-chain composition of glycerolipids. These enzymes compete with fatty acid-modifying enzymes, such as elongases and the fatty acid desaturase. On the other hand, membrane functions are critically determined by the membrane phospholipid composition. We propose that acyl-transferases are organized in functional complexes that allow metabolic channeling of lipid intermediates, such as lyso-phosphatidic acid or diacylglycerol, which are also potent signaling molecules and highly membrane-active compounds. Numerous potential ‘scaffolding proteins’ that may be involved in organizing these functional complexes have been identified in previous screens of mutant collections in our laboratory and are now being explored. Some of these proteins share structural and functional homology to mammalian proteins, known to be involved in regulating lipid droplet formation.
We use a broad spectrum of biochemical, genetic and molecular biological techniques to identify and characterize novel genes and gene products and regulatory processes involved in fatty acid homeostasis and acyl-transfer into membrane and storage lipids. High-resolution live cell imaging is applied to study protein localization and structure, dynamics, interaction and inheritance of organelles. Label-free lipid imaging using Coherent anti-Stokes Raman Scattering (CARS)-microscopy introduces novel and exciting approaches to understand lipid fluxes and lipid homeostasis in living cells and organisms.
Laboratory know-how and infrastructureEstablished methodology includes: yeast genetics and cell biology with all relevant genetic, molecular and bioinformatics techniques. We use yeast DNA arrays for transcriptional profiling of yeast mutants, and large-scale analyses using robotics for phenotypic testing of yeast mutants (SGA analysis). Lipid analysis technology includes automated TLC (total lipids; large scale screenings of lipid mutants); GLC (fatty acids), HPLC (phospho- and neutral lipids), GC/MS (fatty acids, sterols; lipid identification) and nano-ESI tandem MS and qTOF MS (lipid molecular species). Various systems for heterologous expression of mammalian enzymes, for functional analysis and large-scale protein production, are established. The lab is equipped with two confocal laser scanning microscopes, including multi-photon laser setups for CARS and SHG imaging. This infrastructure enables 4D-imaging, bleaching techniques (FRAP, FLIM), FRET, and dynamic metabolic imaging studies using deuterated compounds (CARS). Access to electron microscopy and tomography facilities, including high-pressure freezing is readily available.