Analysis of the membrane topology for transmembrane domains 7-12 of the human reduced folate carrier by scanning cysteine accessibility methods.
The hRFC (human reduced folate carrier) is the major membrane transporter for both reduced folates and antifolates in human tissues and tumours. The primary amino acid sequence of hRFC predicts a membrane topology involving 12 TMDs (transmembrane domains) with cytosolic oriented N- and C-termini, and a large internal loop connecting TMDs 6 and 7. Previous studies using haemagglutinin epitope insertion and scanning glycosylation mutagenesis methods verified portions of the predicted topology model, including TMDs 1-8 and the N- and C-termini of hRFC. However, the topology structure for TMDs 9-12 remains controversial. To further determine the membrane topology of the hRFC protein, single cysteine residues were introduced into the predicted extracellular or cytoplasmic loops of a fully functional cysteine-less hRFC expressed in transport impaired MtxRIIOua(R)2-4 Chinese hamster ovary cells. The membrane orientations of the substituted cysteines were determined by treatments with the thiol reagents 3-(N-maleimidylpropionyl)-biocytin (biotin maleimide) and 4-acetamido-4'maleimidylstilbene-2,2'-disulphonic acid (stilbenedisulphonate maleimide; SM) or N-ethylmaleimide, combined with the cell-permeabilizing reagent SLO (streptolysin O). We found that cysteine residues placed in the predicted extracellular loops between TMDs 7 and 8 (position 301), 9 and 10 (360), and 11 and 12 (429) could be biotinylated with 200 microM biotin maleimide, and labelling could be blocked with SM. However, biotinylation of cysteines placed in the predicted intracellular loops between TMDs 8 and 9 (position 332) and TMDs 10 and 11 (position 388) was only detected after cell permeabilization with SLO and was abolished by pre-treatment with N -ethylmaleimide. These results strongly support a 12-TMD topology structure for the hRFC protein.
Department of Pharmacology, Wayne State University School of Medicine, 540 E. Confield Ave., Detroit, MI 48201, USA.
Wei
W
Cao
Larry H
LH
Matherly
D000818:Animals; D001692:Biological Transport; D019921:Biotinylation; D016466:CHO Cells; D006224:Cricetinae; D003545:Cysteine / Q000032:analysis* / Q000235:genetics; D006801:Humans; D008301:Maleimides / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D058978:Reduced Folate Carrier Protein; D013439:Sulfhydryl Reagents / Q000737:chemistry
D008301:Maleimides; D026901:Membrane Transport Proteins; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; D013439:Sulfhydryl Reagents; C043592:maleimide; D003545:Cysteine