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). ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32DK063750-01
Application #
6584217
Study Section
Special Emphasis Panel (ZRG1-F10 (20))
Program Officer
Podskalny, Judith M,
Project Start
2003-02-04
Project End
2006-02-03
Budget Start
2003-02-04
Budget End
2006-02-03
Support Year
1
Fiscal Year
2003
Total Cost
$56,308
Indirect Cost
Name
University of California Irvine
Department
Physiology
Type
Schools of Medicine
DUNS #
046705849
City
Irvine
State
CA
Country
United States
Zip Code
92697
Subramanian, Veedamali S; Marchant, Jonathan S; Said, Hamid M (2008) Apical membrane targeting and trafficking of the human proton-coupled transporter in polarized epithelia. Am J Physiol Cell Physiol 294:C233-40
Subramanian, Veedamali S; Marchant, Jonathan S; Said, Hamid M (2006) Biotin-responsive basal ganglia disease-linked mutations inhibit thiamine transport via hTHTR2: biotin is not a substrate for hTHTR2. Am J Physiol Cell Physiol 291:C851-9
Subramanian, Veedamali S; Marchant, Jonathan S; Said, Hamid M (2006) Targeting and trafficking of the human thiamine transporter-2 in epithelial cells. J Biol Chem 281:5233-45
Subramanian, Veedamali S; Marchant, Jonathan S; Boulware, Michael J et al. (2004) A C-terminal region dictates the apical plasma membrane targeting of the human sodium-dependent vitamin C transporter-1 in polarized epithelia. J Biol Chem 279:27719-28
Said, Hamid M; Balamurugan, Krishnaswamy; Subramanian, Veedamali S et al. (2004) Expression and functional contribution of hTHTR-2 in thiamin absorption in human intestine. Am J Physiol Gastrointest Liver Physiol 286:G491-8
Subramanian, Veedamali S; Marchant, Jonathan S; Parker, Ian et al. (2003) Cell biology of the human thiamine transporter-1 (hTHTR1). Intracellular trafficking and membrane targeting mechanisms. J Biol Chem 278:3976-84