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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doaj-e004359ac5264826b7fec2baf8f335f22021-03-03T22:49:10ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-01810e7830110.1371/journal.pone.0078301Delineating the extracellular water-accessible surface of the proton-coupled folate transporter.Phaneendra Kumar DuddempudiRaman GoyalSwapneeta Sanjay DateMichaela JansenThe 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.https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24205192/?tool=EBI |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Phaneendra Kumar Duddempudi Raman Goyal Swapneeta Sanjay Date Michaela Jansen |
spellingShingle |
Phaneendra Kumar Duddempudi Raman Goyal Swapneeta Sanjay Date Michaela Jansen Delineating the extracellular water-accessible surface of the proton-coupled folate transporter. PLoS ONE |
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) |
series |
PLoS ONE |
issn |
1932-6203 |
publishDate |
2013-01-01 |
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. |
url |
https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24205192/?tool=EBI |
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