Regulation and Transport Mechanism of Choline Transporter-Like Protein 1 (CTL1/SLC44A1)

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As an essential nutrient, choline is mostly consumed for the biosynthesis of membrane phospholipids. Some choline is oxidised in mitochondria to betaine and only small amount of choline is used for acetylcholine synthesis. Choline is positively charged quaternary amine and requires a protein-mediated transport to pass the membranes. We recently established that choline-transporter-like protein 1 (CTL1) function as a choline transporter at the plasma membrane and mitochondria. CTL1 function depends on cell differentiation state and choline content. Choline deficiency reduces CTL1 levels, causing impairments in the membrane synthesis and increasing accumulation of lipid droplets. Choline addition stimulates CTL1 gene expression and restores its transport function. There are multiple unknowns about the CTL1 regulation and transport mechanism. We plan to elucidate: 1) The nuclear mechanisms that control CTL1 transcription based on cellular demands for choline and during the process of specific cell (muscle and heart ) differentiation. To establish the mechanisms for this widely present and significant regulatory response we will examine the CTL1 gene regulation with both nuclear and epigenetic modulators. We have characterized mammalian CTL1 genes and produced promoter-luciferase reporter constructs for CTL1. We will investigate the CTL1 promoter methylation and specific transcriptional regulation with nuclear factors Sp1, NF1 and E2F for which we know DNA binding elements and function. Other relevant regulators will include muscle and heart differentiation factors MyoD and GATA4. They particular roles will be also carefully examined in the context of choline availability using genome-wide and gene-specific approaches. 2) The membrane transport mechanism of CTL1. We have produced two specific antibodies and established that CTL1 transporter has nine transmembrane domains, intracellular N-terminus and extracellular C-terminus. Yet, little is currently known about the function of specific domains and amino acid residues responsible for CTL1-mediated transport. Of particular importance is to identify which transporter regions are critical for choline transport and for the membrane targeting at the cell surface and mitochondria. The CTL1 membrane position will be confirmed by cysteine scanning. For  determination of substrate binding site (positively charged choline),  focus will be on negatively charged amino acid residues mostly located in the outside area and on the last two transmembrane domains, all found to be the evolutionary conserved in the transporter family SLC44A. We expect to establish how specific structural components govern the CTL1 transport function, oligomerization and sensitivity to inhibition with  specific inhibitor chemicholinium-3.