FLIPPASES · Lipid flippases - Protein-mediated lipid translocation Regulation and physiological significance of transbilayer lipid distribution

Final Activity Report Summary - FLIPPASES (Lipid flippases - Protein-mediated lipid translocation Regulation and physiological significance of transbilayer lipid distribution)

The dynamics of biomembrane lipid organisation and its physiological role is a rapidly developing interdisciplinary field. Membrane dynamics is largely controlled by lipid transporters also known as flippases, which play a pivotal role in cell homeostasis. Putative flippases belong to various subfamilies of the P-type ATPases and ATP-binding cassette (ABC) transporters. These proteins are involved in regulating lipid asymmetry, cellular signalling, in the budding of transport vesicles and protein synthesis. Dysfunction of flippases causes severe diseases like cholestasis, atherosclerosis, visual impairment, what emphasises their crucial physiological function. Although these proteins have been connected with the translocation of various classes of membrane lipids across cellular membranes, biochemical and genetic proof of their function and the key features of their activity remain to be elucidated. The network teaming 12 groups was aimed at undertaking an interdisciplinary effort to reveal the molecular identity and cellular localisation of flippases, their three-dimensional structure, substrate specificity, the molecular mechanism, energetics and regulation of their functioning, as well as unravelling the consequences of their action for cell and body physiology. Biophysical approaches have been used to address lipid transport energetics and its role in membrane budding, fission and fusion. 23 young researchers recruited by the network were immediate protagonists of network research thereby receiving high quality training not only in research, but also in international project management. Intense collaborations established a network gathering complementary expertise at the highest standard in all required techniques of molecular and cell biology, biochemistry, proteomics, structural biology including X-ray crystal analysis and cryoelectron microscopy, and theoretical and experimental biophysics. As a result new membrane proteins involved in lipid transport and metabolism were discovered and characterised with respect to structure, localisation, substrate specificity, consequences for other processes of the cell and for structure and dynamics of respective membranes. Importantly, the network has shown that lipid transporters play, pivotal roles in mammalian, plant and other eukaryotic cells like yeast. For example, P-type-ATPases transport lipid in parasites as Leishmania. On the same time they transport antileishmania drugs, an important outcome for understanding drug resistance. Another example is the evidence for a lipid-transport independent function of a P-type-ATPase in the proper assembly of the apical brush border in human intestinal epithelial cells which discovered by the network. This function may be relevant for the clinical manifestation of familial intrahepatic cholestasis, a life threatening liver disease. Other milestones of the project are development of high yield expression systems for lipid transporters enabling the in-depth characterisation of their activity as well as three dimension structure determination. First structure determinations of those proteins were already successful. In addition to providing novel applicative insights into flippase-related diseases, the research of the network was and is essential for identifying potential targets for designed drug development in pharmaceutics, and will offer new biotechnological tools to use cellular model systems in production of lipid transporters and lipophilic drugs. The network succeeded to strengthen interaction between experiment and theory in research and developed models on which basis experimental results can be interpreted in a quantitative manner. The network organised two international conferences related to lipid transporters. By that it established a European wide network in this field acting beyond the funding period. Finally, a text book on lipid transporters with experts from all over the world will appear in 2009.

Data: CORDIS, © European Union