Topological and functional analysis of the human reduced folate carrier by hemagglutinin epitope insertion.
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.
Ferguson
P L
PL
Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 5C1, Canada.
Flintoff
W F
WF
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D002352:Carrier Proteins / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry*; D006224:Cricetinae; D017931:DNA Primers; D000939:Epitopes; D005492:Folic Acid / Q000737:chemistry*; D006031:Glycosylation; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D058978:Reduced Folate Carrier Protein; D013329:Structure-Activity Relationship; D014162:Transfection
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D017931:DNA Primers; D000939:Epitopes; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; C120043:SLC19A2 protein, human; D005492:Folic Acid