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.
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.
S C
SC
Wong
L
L
Zhang
S A
SA
Proefke
L H
LH
Matherly
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