Membrane topological analysis of the proton-coupled folate transporter (PCFT-SLC46A1) by the substituted cysteine accessibility method.
The proton-coupled folate transporter (PCFT) mediates intestinal folate absorption. Loss-of-function mutations in this gene are the molecular basis for the autosomal recessive disorder, hereditary folate malabsorption. In this study, the substituted cysteine accessibility method was utilized to localize extra- or intracellular loops connecting predicted PCFT transmembrane domains. Cysteine-less PCFT was generated by replacement of all seven cysteine residues with serine and was shown to be functional, following which cysteine residues were introduced into predicted loops. HeLa cells, transiently transfected with these PCFT mutants, were then labeled with an impermeant, cysteine-specific biotinylation reagent (MTSEA-biotin) with or without permeabilization of cells. The biotinylated proteins were precipitated by streptavidin beads and assessed by Western blotting analysis. The biotinylation of PCFT was further confirmed by blocking cysteine residues with impermeant 2-sulfonatoethyl methanethiosulfonate. Two extracellular cysteine residues (66, 298) present in WT-PCFT were not biotinylated; however, in the absence of either one, biotinylation occurred. Likewise, biotinylation occurred after treatment with beta-mercaptoethanol. Taken together, these analyses establish a PCFT secondary structure of 12 transmembrane domains with the N- and C- termini directed to the cytoplasm. The data indicate further that there is a disulfide bridge, which is not required for function, between the native C66 and C298 residues in the first and fourth transmembrane domains, respectively.
Zhao
Rongbao
R
Department of Medicine, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Unal
Ersin Selcuk
ES
Shin
Daniel Sanghoon
DS
Goldman
I David
ID
D019921:Biotinylation; D003545:Cysteine; D004220:Disulfides; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry*; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D015336:Molecular Probe Techniques; D010449:Peptide Mapping; D011485:Protein Binding; D017433:Protein Structure, Secondary; D058979:Proton-Coupled Folate Transporter
D004220:Disulfides; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D058979:Proton-Coupled Folate Transporter; C505058:SLC46A1 protein, human; D003545:Cysteine