Thiamine (vitamin B1) is an important nutrient that is essential for human health. Recently two thiamine transporter genes SLC19A2 and SLC19A3 (encoding hTHTR1 and hTHTR2, respectively) have been cloned and characterized from a number of human tissues. Nothing is known about the membrane targeting and intracellular trafficking of these thiamine transporters. Targeting of a protein to the plasma membrane has been shown to involve specific targeting signals such as tyrosine, di-leucine, diacidic, dibasic, histidine, and PDZ domains encoded in the polypeptide. The hTHTRI& 2 polypeptides have a number of such potential sorting signals. In preliminary studies, we fused hTHTR1 to EGFP and demonstrated functionality and membrane localization of the stably expressed fusion protein (hTHTR1-EGFP) in HuTu-80 cells. In order to investigate the role of specific targeting signals in directing hTHTRI& 2 to the plasma membrane of human intestinal epithelial cells, I will compare wild-type, truncated, and site-directed mutant constructs of hTHTRI& 2 fused to EGFP, and image by confocal microscopy.
I aim to also determine if the expression of hTHTRI& 2 at a given cell membrane domain is cell-type specific using wild-type constructs of hTHTRI& 2 proteins fused to EGFP. To further examine the intracellular trafficking of hTHTRI& 2 proteins I will examine the role of the microtubule network and the actin-based cytoskeleton, perform kinetic analysis of the delivery of newly synthesized thiamine transporters to the plasma membrane, and investigate the role of vesicular transport of the proteins to the plasma membrane (insertion/retrival). ? ?
