Role of the C-terminus and the long cytoplasmic loop in reduced folate carrier expression and function.
The reduced folate carrier (RFC1), a member of the major facilitative superfamily, generates uphill transport of folates into cells through an exchange mechanism with intracellular organic anions. RFC1 has twelve transmembrane domains with N- and C-termini, and the long loop connecting the 6th and 7th transmembrane domains, directed to the cytoplasm. To elucidate the role of the C-terminus and the long cytoplasmic loop in carrier function, mutants with deletion of the entire C-terminus or with progressive deletions of the loop region were constructed and stably transfected into the murine MTX(r)A cell line, which lacks functional RFC1. While expression of the C-terminus-deleted RFC1 protein could not be detected in the cell lysate, the RFC1 mutant lacking 57 of 66 amino acid residues of the long cytoplasmic loop appeared to be inserted into the cytoplasmic membrane but was not functional. In cell lines in which 17 or 31 amino acids were deleted from the carboxyl half of the loop, there was partial preservation of methotrexate, 5-formyltetrahydrofolate, and 5-methyltetrahydrofolate transport. The loss of 5-formyltetrahydrofolate transport activity in the delta31 and delta17 mutants was due primarily to a decrease in substrate binding to the carrier. Mutants with partially truncated internal loops demonstrated an anion responsiveness similar to that of wild-type RFC1, indicating that this region of the carrier does not contain a site(s) that plays a role in anion exchange. This is the first study to describe the important role of the long cytoplasmic loop in substrate binding and the crucial role of the C-terminus in maintaining stability of RFC1.
Sharina
Iraida G
IG
Department of Medicine, Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Zhao
Rongbao
R
Wang
Yanhua
Y
Babani
Solomon
S
Goldman
I David
ID
D000595:Amino Acid Sequence; D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000378:metabolism / Q000494:pharmacology; D001692:Biological Transport; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D017353:Gene Deletion; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D026901:Membrane Transport Proteins; D008727:Methotrexate / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D000964:Antimetabolites, Antineoplastic; D002352:Carrier Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C492292:Slc19a2 protein, mouse; D008727:Methotrexate