Functional role of arginine 373 in substrate translocation by the reduced folate carrier.
The reduced folate carrier (RFC) plays a critical role in the cellular uptake of folates. However, little is known regarding the mechanism used to transport substrates or the tertiary structure of the protein. Through the analysis of a Chinese hamster ovary cell line deficient in folate uptake, we have identified a single residue in TM10 (Arg-373) of RFC that appears to play a critical role in the translocation of substrate. Replacement of this position with various amino acids (KHQNA) diminished the rate of translocation by 16-50-fold, although substrate binding, protein stability, and localization were unaffected. Furthermore, the translocation capabilities of an R373C mutant in a cysteine-less form of the reduced folate carrier were enhanced 2.5-fold by the positively charged methanethiosulfonate reagent, confirming the essential role of a positive charge at this position. When considering the membrane-impermeable nature of this reagent, the data further suggest that the Arg-373 residue is located within the substrate translocation pathway of the RFC protein. Moreover, cross-linking analysis of the Arg-373 residue demonstrates that it is within 6 A of residue Glu-394 (TM11), providing the first definitive tertiary structural information for this protein.
Sadlish
Heather
H
Department of Microbiology and Immunology, University of Western Ontario, London, Canada.
Williams
Frederick M R
FM
Flintoff
Wayne F
WF
D000818:Animals; D001120:Arginine; D001692:Biological Transport; D016466:CHO Cells; D002352:Carrier Proteins / Q000737:chemistry* / Q000502:physiology; D006224:Cricetinae; D026901:Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D058978:Reduced Folate Carrier Protein; D013329:Structure-Activity Relationship
D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D058978:Reduced Folate Carrier Protein; D001120:Arginine