Effects of the loss of capacity for N-glycosylation on the transport activity and cellular localization of the human reduced folate carrier.
The role of N-glycosylation in reduced folate carrier (RFC) transport and membrane targeting was examined in transport-deficient K562 (K500E) cells transfected with human RFC cDNAs. Treatment of cells expressing wild-type RFC with tunicamycin (0-3 microg) resulted in a progressive shift of the approximately 85 kDa RFC on western blots to 65 kDa. At 3 microg/ml tunicamycin, the nearly complete loss of glycosylated RFC was accompanied by a approximately 25% decreased rate of methotrexate uptake. A deglycosylated RFC cDNA construct in which asparagine-58 was replaced by glutamine (Gln58-RFC) was expressed in K500E cells as a 65 kDa protein and restored transport capacity for methotrexate and (6S)5-formyl tetrahydrofolate. With both wild-type and Gln58-RFC constructs, expression of cDNA-encoded RFC protein far exceeded relative levels of RFC uptake. Wild-type and Gln58-RFCs containing a hemagglutinin (HA) epitope at the carboxyl terminus were similarly functional and, by immunofluorescence staining with rhodamine-conjugated anti-HA antibody, were localized to plasma membranes. Collectively, our results demonstrate that N-glycosylation of human RFC plays no significant role in either transport function or membrane targeting. The discrepancy between the stoichiometries of RFC expression and transport activity for both wild-type RFC and Gln58-RFC implies that identical regulatory controls and/or non-RFC transport components are necessary to completely restore transport function in the transfected cells.
Wong
S C
SC
Experimental and Clinical Therapeutics Program, Barbara Ann Karmanos Cancer Institute and the Department of Pharmacology, School of Medicine, Wayne State University, 110 E. Warren Ave., Detroit, MI 48201, USA.
Zhang
L
L
Proefke
S A
SA
Matherly
L H
LH
D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005492:Folic Acid / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D020014:K562 Cells; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D058978:Reduced Folate Carrier Protein
D002352:Carrier Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; C120043:SLC19A2 protein, human; D005492:Folic Acid