Characteristics and novel functions of SLC44A transporters

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Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are membrane phospholipids involved in the fundamental processes of cell division, survival and signaling. PC and PE are synthesized de novo by the CDP-choline and CDP-ethanolamine (Kennedy) pathway, in which the extracellular substrates choline and ethanolamine are transported into the cell, phosphorylated, and coupled with diacylglycerol to form the final phospholipid product. While multiple transport systems have been established for choline, ethanolamine transport is poorly characterized, and there is no single protein assigned a transport function for ethanolamine. We investigate transporters from the family of Solute Carriers 44A (SLC44A) known as Choline Transporter-Like proteins-1 and -2 (CTL1 and CTL2). CTL1/SLC44A1 is a choline/proton antiporter and delivers choline for the CDP-choline pathway and choline oxidation to betaine. There are indications that CTL2/SLC44A2 is also a choline transporter at the plasma membrane and mitochondria, but its function is not firmly established. We propose that i. CTL1 and CTL2 are authentic plasma membrane and mitochondria ethanolamine transporters -We will characterize cellular and mitochondrial transport of CTL1 and CTl2 in cells that maintain CTL1 but lack CTL2 and vice versa. We will employ CTL1/2 mutants, siRNA and specific antibodies to demonstrate for the first time that CTL1 and CTL2 are choline and also ethanolamine transporters in mammalian systems. ii. CTL1 and CTL2 regulate mitochondrial function-Our preliminary data suggest that the luck of CTL1 induces mitophagy and fragmentation of mitochondrial networks. We propose that CTL1 regulate mitochondrial fusion-fission processes. We will investigate mechanisms of mitochondrial fusion and metabolism (electron transport complexes, ATP synthesis, ROS production, membrane potential, etc.) in CTL1 and CTL2 deficient and over-expressing cells. The initial findings will be corroborated in mitochondria and primary cells isolated from CTL1 and/or CTL2 knockout mice. We expect to demonstrate for the first time that CTL1 and perhaps CTL2 are critical regulators of mitochondrial turnover and mitochondrial energy homeostasis. iii. CTL1 and CTL2 couple phospholipid synthesis with the whole-body energy metabolism-CTL1 and CTL2 knockout mice were produced and characterized by the International Mouse Phenotyping Consortium. Reduced body weight and increased muscle/fat ratio are common features of CTL1 and CTL2 knockout mice. We will characterize CTL1 mice in full detail and compare to better known CTL2 mice, including the regulation of the  Kennedy pathway, lipid synthesis and energy metabolism in skeletal muscle, liver and adipose tissue, applying protocols extensively used in previous studies of our Pcyt2 knockout mice (Pcyt2 is the main regulatory enzyme in the CDP-ethanolamine Kennedy pathway).