Choline transporter-like proteins 1 and 2 are newly identified plasma membrane and mitochondrial ethanolamine transporters

Phosphatidylcholine and phosphatidylethanolamine are membrane phospholipids considered important components of cellular membranes, as they are involved with essential cellular processes. The de novo synthesis of phosphatidylcholine and phosphatidylethanolamine is by the CDP-choline and CDP-ethanolamine branches of the Kennedy pathway. In this pathway, choline and ethanolamine, which are extracellular substrates are actively transported into the cell where they undergo phosphorylation, and are then coupled with diacylglycerols to form a phospholipid product. Several transport systems have been proposed for choline. Specifically, solute carriers known as choline transporter-like proteins-1 and -2 (CTL1 and CTL2) have been identified as choline transporters at the plasma membrane and mitochondria. In contrast, ethanolamine transport has been poorly characterized with the absence of a single gene/protein assigned a transport function for mammalian ethanolamine. However, there have been early kinetic studies in bovine endothelial cells, human retinoblastoma cells, and glial cells that have implicated that mammalian ethanolamine and choline transport may share a similar transport system.

In a new study published in the Journal of Biological Chemistry, Adrian Taylor, Sophie Grapentine, Jasmine Ichhpuniani and Professor Marica Bakovic all from the Department of Human Health and Nutritional Sciences at the University of Guelph in Canada proposed that CTL1 was a transporter for ethanolamine/choline and the last missing link between CDP-choline and CDP-ethanolamine pathways for phospholipids synthesis. They therefore sought to examine the kinetics of ethanolamine transport in both CTL1 and CTL2 depleted and overexpressing cells. Their results showed that CTL1 and CTL2 are involved in ethanolamine transport at both the cell surface and mitochondria.

Through a series of molecular and functional experiments, the research team found that ethanolamine was a substrate for CTL1- and CTL2-mediated transports and that the inhibition of CTL1 and CTL2 led to a reduction in ethanolamine and choline transport. CTL1 was found to be a high-affinity ethanolamine transporter, while CTL2 was a low-affinity transporter. The affinity constant of CTL1  for ethanolamine binding was found to be in the range of physiological ethanolamine concentration in rat and humans. In addition, they also found that CTL1 deficiency downregulated the CDP-Ethanolamine Kennedy pathway. Furthermore, CTL1 and CTL2 independently contributed to both the CDP-Ethanolamine Kennedy pathway and CDP- choline. In CTL1-deficient cells, CTL2 was not overexpressed.

When the authors overexpressed CTL1 and CTL2 proteins, it facilitated choline and ethanolamine transport, with CTL1 and CTL2 displaying similar affinities for choline and ethanolamine. The overexpression of CTL1 in CTL1-deficient cells facilitated the CDP-Ethanolamine Kennedy pathway by increasing the flux through the CDP-Ethanolamine pathway in CTL1-deficient cells.

CTL1, the high-affinity transporter transported choline and ethanolamine similarly. Whereas, in the low-affinity transporter CTL2, the affinity for choline and ethanolamine was different and reduced with more preference for ethanolamine as its substrate. CTL1 and CTL2 mediated mitochondrial ethanolamine transport as well as in the whole cells with the same kinetic properties. However, in both the whole cells and mitochondria, CTL1 contributed the most of the ethanolamine transport.

In summary, the authors have identified a novel function of CTL1 and CTL2 in the transport of ethanolamine. The study is the first to show that CTL1 and CTL2 are the physiological transporters for ethanolamine in the mitochondria and whole cells. This is also the first study to demonstrate the intrinsic roles of CTL1 and CTL2 in the de novo synthesis of phosphatidylethanolamine by the CDP-Ethanolamine Kennedy pathway and intracellular compartmentation of ethanolamine. The new findings will help develop new prevention and treatment strategies in various diseases associated with mutations in CTL1 and CTL2 genes.