Intestinal folate transport: identification of a cDNA involved in folate transport and the functional expression and distribution of its mRNA.
Although the mechanism of folate intestinal transport has been the subject of intensive studies, very little is known about the molecular identity of the transport system(s) involved. In this investigation, we screened a mouse intestinal cDNA library using as probe the cDNA clone of a reduced folate carrier (RFC1) of mouse leukemia L1210 cells, and identified a positive clone, IFC1(RFC1). The cloned cDNA consisted of 2274 base pairs with an open reading frame that encodes a putative polypeptide of 512 amino acids with a predicted molecular mass of 58,112 daltons and 12 putative transmembrane domains. The polypeptide appears to carry a net positive charge (pI = 8.6) which may be important for its interaction with the negatively charged substrate. Functional identity of the IFC1(RFC1) clone was established by expression in Xenopus oocytes. An 11-fold increase in 5-methyltetrahydrofolate (5-MTHF) uptake was observed in oocytes injected with 10 ng IFC1(RFC1) cRNA compared to water-injected controls. The expressed folate uptake in the cRNA injected oocyte was (1) 4,4'-diisothiocyanatosilbene-2,2'-disulfonic acid (DIDS)-sensitive; and (2) saturable with an apparent Km of 1.99 +/- 0.32 micrometers and a V(max) of 3782 +/- 188 fmol/oocyte per h. The distribution of mRNA species complementary to IFC1(RFC1) in different mouse tissues was examined by Northern blot analysis. In addition to the small intestine, expression of such mRNA species were also found in the kidney, large intestine, brain, heart and liver. Furthermore, mRNA species complementary to IFC1(RFC1) were also detected by Northern blot analysis in the small intestine of human and other animal species (rat and rabbit). Expression of mRNA complementary to IFC1(RFC1) was markedly higher in rat intestinal villus cells than in crypt cells. These results represent the first identification of a folate transporter in mammalian intestine.
Said
H M
HM
VA Medical Center - 151, Long Beach, California 90822, USA.
Nguyen
T T
TT
Dyer
D L
DL
Cowan
K H
KH
Rubin
S A
SA
D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D002460:Cell Line; D018076:DNA, Complementary / Q000032:analysis*; D005260:Female; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D007408:Intestinal Absorption; D007939:Leukemia L1210; D051379:Mice; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D011956:Receptors, Cell Surface; D017422:Sequence Analysis, DNA; D014982:Xenopus laevis
D002352:Carrier Proteins; D018076:DNA, Complementary; D058973:Folate Receptors, GPI-Anchored; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D005492:Folic Acid