Delineating the extracellular water-accessible surface of the proton-coupled folate transporter.

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 usi...

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Autores principales:	Phaneendra Kumar Duddempudi, Raman Goyal, Swapneeta Sanjay Date, Michaela Jansen
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/e004359ac5264826b7fec2baf8f335f2
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spelling	oai:doaj.org-article:e004359ac5264826b7fec2baf8f335f22021-11-18T08:50:17ZDelineating the extracellular water-accessible surface of the proton-coupled folate transporter.1932-620310.1371/journal.pone.0078301https://doaj.org/article/e004359ac5264826b7fec2baf8f335f22013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24205192/?tool=EBIhttps://doaj.org/toc/1932-6203The 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.Phaneendra Kumar DuddempudiRaman GoyalSwapneeta Sanjay DateMichaela JansenPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 10, p e78301 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Phaneendra Kumar Duddempudi Raman Goyal Swapneeta Sanjay Date Michaela Jansen Delineating the extracellular water-accessible surface of the proton-coupled folate transporter.
description	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.
format	article
author	Phaneendra Kumar Duddempudi Raman Goyal Swapneeta Sanjay Date Michaela Jansen
author_facet	Phaneendra Kumar Duddempudi Raman Goyal Swapneeta Sanjay Date Michaela Jansen
author_sort	Phaneendra Kumar Duddempudi
title	Delineating the extracellular water-accessible surface of the proton-coupled folate transporter.
title_short	Delineating the extracellular water-accessible surface of the proton-coupled folate transporter.
title_full	Delineating the extracellular water-accessible surface of the proton-coupled folate transporter.
title_fullStr	Delineating the extracellular water-accessible surface of the proton-coupled folate transporter.
title_full_unstemmed	Delineating the extracellular water-accessible surface of the proton-coupled folate transporter.
title_sort	delineating the extracellular water-accessible surface of the proton-coupled folate transporter.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/e004359ac5264826b7fec2baf8f335f2
work_keys_str_mv	AT phaneendrakumarduddempudi delineatingtheextracellularwateraccessiblesurfaceoftheprotoncoupledfolatetransporter AT ramangoyal delineatingtheextracellularwateraccessiblesurfaceoftheprotoncoupledfolatetransporter AT swapneetasanjaydate delineatingtheextracellularwateraccessiblesurfaceoftheprotoncoupledfolatetransporter AT michaelajansen delineatingtheextracellularwateraccessiblesurfaceoftheprotoncoupledfolatetransporter
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Delineating the extracellular water-accessible surface of the proton-coupled folate transporter.

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