| Choline is an essential nutrient required for the synthesis of the neurotransmitter acetylcholine and the major phospholipid of mammalian membranes, phosphatidylcholine. Choline is also oxidized in the mitochondria to betaine, a methyl group donor in homocysteine methylation and a vital organic osmolyte. The solute carrier 44A1 (SLC44A1) is a recently discovered choline transport protein with an intermediate affinity for choline, that is ubiquitously expressed and mediates a Na+-independent, hemicholinium-3 sensitive, choline transport. The present study was initiated by the successful development of two specific SLC44A1 antibodies, and establishment of ideal conditions for immunocytochemistry and immunoblotting experiments. A topology model for SLC44A1 was developed, putative phosphorylation sites and regulatory mechanisms identified, and subcellular localization analyzed. Endogenous and over-expressed SLC44A1 localization was discovered in the outer membrane of mouse and human mitochondria using confocal microscopy, differential centrifugation and Western blotting. Targeted knock-down of SLC44A1 using siRNA technology caused the disappearance of the protein from the mitochondrial membrane. Choline uptake competition studies on isolated mitochondria using hemicholinium-3 and SLC44A1 antibodies significantly decreased mitochondrial choline uptake, while the uptake was increased by overexpression of SLC44A1. These data clearly establish SLC44A1 as a mitochondrial choline transport protein.;This thesis provides the foundation for establishing the metabolic role of the choline transporter, SLC44A1, by developing tools for the analysis of this protein and identifying its localization and function in the mitochondrial membrane.;During choline deficiency, SLC44A1 mRNA expression was found to be downregulated in mouse liver and muscle cells, but SLC44A1 protein was only reduced in muscle cells. As expected, choline uptake was significantly decreased across the mitochondrial membrane of choline deficient muscle cells. There was a dramatic decrease in phosphatidylcholine synthesis and an increase in lipid droplet formation in both cell types under choline deficiency conditions, resulting from diacylglycerol esterification with preexisting fatty acids. Studies of metabolic consequences of impaired SLC44A1 function in vivo were also initiated. We describe here the phenotype of a patient with impaired betaine synthesis, as well as the development of a SLC44A1-deficient mouse germline. |
