Cloning and functional characterization of a folate transporter from the nematode Caenorhabditis elegans.
Two putative orthologs to the human reduced folate carrier (hRFC), folt-1 and folt-2, which share a 40 and 31% identity, respectively, with the hRFC sequence, have been identified in the Caenorhabditis elegans genome. Functional characterization of the open reading frame of the putative folt-1 and folt-2 showed 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 K(m) of 1.23 +/- 0.18 microM), a similar degree of inhibition by reduced and substituted folate derivatives, sensitivity to the anti-inflammatory drug sulfasalazine (apparent K(i) of 0.13 mM), and inhibition by anion transport inhibitors, e.g., DIDS. Knocking down (silencing) or knocking out the folt-1 gene led to a significant inhibition of folate uptake by intact living C. elegans. We also cloned the 5'-regulatory region of the folt-1 gene and confirmed promoter activity of the construct in vivo in living C. elegans. With the use of 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. Furthermore, 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 the folate uptake process of the human intestine. Thus C. elegans provides a genetically tractable model that can be used to study integrative aspects of the folate uptake process in the context of the whole animal level.
Balamurugan
Krishnaswamy
K
VA Medical Center-151, Long Beach, CA 90822, USA.
Ashokkumar
Balasubramaniem
B
Moussaif
Mustapha
M
Sze
Ji Ying
JY
Said
Hamid M
HM
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D017173:Caenorhabditis elegans / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D005492:Folic Acid / Q000031:analogs & derivatives / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D020868:Gene Silencing; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011487:Protein Conformation; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D058978:Reduced Folate Carrier Protein; D012964:Sodium / Q000378:metabolism; D012460:Sulfasalazine / Q000494:pharmacology; D014162:Transfection
D000894:Anti-Inflammatory Agents, Non-Steroidal; D029742:Caenorhabditis elegans Proteins; D026901:Membrane Transport Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D012460:Sulfasalazine; D005492:Folic Acid; D012964:Sodium; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid