Abstract
The membrane topology of the human reduced folate carrier protein (591 amino acids) was assessed by single insertions of the hemagglutinin epitope into nine sites of the protein. Reduced folate carrier-deficient Chinese hamster ovary cells expressing each of these constructs were probed with anti-hemagglutinin epitope monoclonal antibodies to assess whether the insertion was exposed to the external environment or to the cytoplasm. The results are consistent with the 12-transmembrane topology predicted for this protein. The hemagglutinin epitope insertion mutants were also tested for their effects on the function of the reduced folate carrier. For these studies, each of the constructs had a carboxyl-terminal fusion of the enhanced green fluorescent protein to monitor and quantitate expression. Insertions into the external loop between transmembrane regions 7 and 8 (Pro-297), the cytoplasmic loop between transmembrane regions 6 and 7 (Ser-225), and near the cytoplasmic amino and carboxyl termini (Pro-20 and Gly-492, respectively) had minor effects on methotrexate binding and uptake. The insertion into the cytoplasmic loop between transmembrane regions 10 and 11 (Gln-385) greatly reduced both binding and uptake of methotrexate, whereas the insertion into the external loop between transmembrane regions 11 and 12 (Pro-427) selectively interfered with uptake but not binding.
Highlights
Mammalian cells require reduced folates for several biochemical pathways involving purine, pyrimidine, and amino acid metabolism
In the first approach, based on that described by Canfield and Levenson [34], the cDNA was digested at unique restriction endonuclease sites (ApaI, 254; NotI, 883; CpoI, 1374; StuI, 1570; numbering based on the human rfc cDNA reported in Ref. 16) predicted to be in intra- or extracellular loops
There was no significant difference in numbers of colonies obtained for these four constructs, indicating that neither the 94 base pairs of the rfc 5Ј-UTR nor the enhanced green fluorescent protein (EGFP) fusion affect the ability of the transfected reduced folate carrier (RFC) to complement functionally the RFC-deficient cell line (Table III)
Summary
Stable clones of the HA insertion constructs in either the 5Ј⌬RFC or 5Ј⌬RFC-EGFP background (described below) were selected and maintained in folic acid-free medium containing 10% dialyzed fetal bovine serum and 2 nM folinic acid [45]. In the first approach, based on that described by Canfield and Levenson [34], the cDNA was digested at unique restriction endonuclease sites (ApaI, 254; NotI, 883; CpoI, 1374; StuI, 1570; numbering based on the human rfc cDNA reported in Ref. 16) predicted to be in intra- or extracellular loops These were blunt-ended using either nuclease S1 or the Klenow fragment of DNA polymerase I, as appropriate to maintain reading frame, and treated with calf intestinal alkaline phosphatase. The underlined nucleotides of the HA insertion primers represent the coding region for the HA epitope
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