title: Delineating the extracellular water-accessible surface of the proton-coupled folate transporter.
abstract: The proton-coupled folate transporter (PCFT) was recently identified as the major uptake route for dietary folates in humans. The three-dimensional structure of PCFT and its detailed interplay with function remain to be determined. We screened the water-accessible extracellular surface of HsPCFT using the substituted-cysteine accessibility method, to investigate the boundaries between the water-accessible surface and inaccessible buried protein segments. Single-cysteines, engineered individually at 40 positions in a functional cysteine-less HsPCFT background construct, were probed for plasma-membrane expression in Xenopus oocytes with a bilayer-impermeant primary-amine-reactive biotinylating agent (sulfosuccinimidyl 6-(biotinamido) hexanoate), and additionally for water-accessibility of the respective engineered cysteine with the sulfhydryl-selective biotinylating agent 2-((biotinoyl)amino)ethyl methanethiosulfonate. The ratio between Cys-selective over amine-selective labeling was further used to evaluate three-dimensional models of HsPCFT generated by homology / threading modeling. The closest homologues of HsPCFT with a known experimentally-determined three-dimensional structure are all members of one of the largest membrane protein super-families, the major facilitator superfamily (MFS). The low sequence identity--14% or less--between HsPCFT and these templates necessitates experiment-based evaluation and model refinement of homology/threading models. With the present set of single-cysteine accessibilities, the models based on GlpT and PepTSt are most promising for further refinement.
authors.affiliation: Department of Pharmacology and Neuroscience, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas, United States of America ; Center for Membrane Protein Research, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas, United States of America.
authors.forename: Phaneendra Kumar
authors.initials: PK
authors.lastname: Duddempudi
authors.forename: Raman
authors.initials: R
authors.lastname: Goyal
authors.forename: Swapneeta Sanjay
authors.initials: SS
authors.lastname: Date
authors.forename: Michaela
authors.initials: M
authors.lastname: Jansen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019921:Biotinylation / Q000379:methods; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D005260:Female; D008565:Membrane Proteins / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D058979:Proton-Coupled Folate Transporter / Q000378:metabolism*; D014867:Water / Q000378:metabolism*; D014982:Xenopus laevis / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D058979:Proton-Coupled Folate Transporter; D014867:Water; D003545:Cysteine