Cloning and functional characterization of a folate transporter from the nematode Caenorhabditis elegans

Krishnaswamy Balamurugan, Balasubramaniem Ashokkumar, Mustapha Moussaif, Ying Sze Ji, Hamid M. Said

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Abstract

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 Km of 1.23 ± 0.18 μM), a 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 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.

Original languageEnglish (US)
Pages (from-to)C670-C681
JournalAmerican Journal of Physiology - Cell Physiology
Volume293
Issue number2
DOIs
StatePublished - Aug 1 2007
Externally publishedYes

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Keywords

  • Folate transport
  • Integrative transport physiology
  • Transport regulation

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

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