Submitted on October 4, 2006
Accepted on April 27, 2007
Cloning and functional characterization of a folate transporter from the nematode, Caenorhabditis elegans
Krishnaswamy Balamurugan1, Balasubramaniem Ashokkumar2, Mustapha Moussaif3, Ji Ying Sze4, and Hamid M. Said1*
1 Medicine (Nephrology)/Physiology/Biophysics, VA Medical Center, Long Beach, CA and University of California, Irvine, California, United States
2 Medicine (Nephrology) & Physiology/Biophysics, VA Medical Center, Long Beach, CA and University of California, Irvine, California, United States
3 Anatomy and Neurobiology, University of California, Irvine, California, United States
4 Irvine, California, United States; Anatomy and Neurobiology, University of California, Irvine, California, United States
* To whom correspondence should be addressed. E-mail: hmsaid{at}uci.edu.
Two putative orthologs to the human reduced folate carrier (hRFC) folt-1 and folt-2 that share a 40% and 31% identity, respectively, with the hRFC sequence have been identified in the C. elegans genome. Functional characterization of the open reading frame of putative folt-1 and folt-2 showed the folt-1 to be a specific folate transporter. Transport of folate by folt-1 expressed in a heterologous expression system showed an acidic pH-dependence, saturability (apparent Km of 1.23 ± 0.18 µM), similar degree of inhibition by reduced and substituted folate derivatives, sensitivity to the anti-inflammatory drug sulfasalazine (apparent Ki of 0.13 mM), and inhibition by the anion transport inhibitors, e.g., 4,4'-diisothio-cyanatostilbene-2,2'-disulphonic acid (DIDS). Knocking down or knocking out the folt-1 gene led to a significant inhibition in folate uptake by intact living C. elegans. We also cloned the 5'-regulatory region of folt-1 gene, and confirmed promoter activity of the construct in vivo in living C. elegans. Using the transcriptional fusion construct (i.e., folt-1::GFP), the expression pattern of folt-1 in different tissues of living animal was found to be highest in the pharynx and intestine. Also, folt-1::GFP expression was developmentally and adaptively regulated in vivo. These studies demonstrate for the first time the existence of a specialized folate uptake system in C. elegans that has similar characteristics to that of folate uptake process of the human intestine. Thus, C. elegan provides a genetically tractable model that can be used for integrative aspects of the folate uptake process in the context of the whole animal level.
