The physiological and molecular function of choline transport in health and metabolic disease

Project Summary Nutrient homeostasis in most living cells is mediated by membrane carrier proteins, which facilitate the translocation of small molecule metabolites across cellular membranes. Despite their clear roles in physiology and disease, many of the small molecule carriers in mammals are poorly studied owing to their hydrophobicity.

Indeed, approximately 30% of these carriers still do not have known substrates or physiological functions. Among small molecule metabolites, choline is a vitamin-like metabolite that is indispensable for cellular and organismal viability. Choline is a dietary component that is critical for the structural integrity of cell membranes, one carbon

metabolism, signaling, cholinergic neurotransmission, and lipid and cholesterol transport and metabolism. Most human cells need to import choline from their extracellular environment. Since serum choline concentration is ~10µM in mammals, choline uptake should occur almost exclusively through high affinity plasma membrane

transporters. However, the identity of the high affinity choline transporter ubiquitously expressed across mammalian tissues remains to be discovered. To address this, in our preliminary work, we used a genome-wide association study (GWAS) of plasma metabolites from a cohort of Finnish individuals and linked biochemical pathways to uncharacterized membrane

transporter genes. This analysis identified a ubiquitously expressed plasma membrane transporter, feline leukemia virus subgroup C cellular receptor 1 (FLVCR1), as a genetic determinant of phosphocholine and phosphatidylcholine levels in human plasma. Biochemical characterization of cells lacking FLVCR1 revealed

striking defects in choline metabolism. Additionally, FLVCR1 loss impairs proliferation of cells under choline limitation. Building upon this observation, in this proposal, we will test the hypothesis that FLVCR1 and its close paralog FLVCR2 are required for choline transport and homeostasis in mammals. To address this, we will first

investigate how loss of FLVCR1-mediated choline import impacts mammalian cell metabolism and physiology. In the second aim, we propose to enhance our understanding of how FLVCR1 and FLVCR2 facilitate choline transport using biochemical and structural studies. Specifically, we will determine structures of FLVCR1/2 in a

ligand-free condition to visualize the conformational changes associated with ligand-binding and release and use mutagenesis to probe the role of residues that directly coordinate choline and those associated with disease. Finally, we will determine the role of FLVCR1-mediated choline import in tissue physiology. In particular, given

the role of choline in liver metabolism, we will focus on the impact of FLVCR1 loss in liver metabolism and non- alcoholic fatty liver disease (NAFLD).