N-linked glycosylation and its impact on the electrophoretic mobility and function of the human proton-coupled folate transporter (HsPCFT).

Biochimica Et Biophysica Acta
Ersin Selcuk UnalI D Goldman

Abstract

The human proton-coupled folate transporter (HsPCFT, SLC46A1) mediates intestinal absorption of folates and transport of folates into the liver, brain and other tissues. On Western blot, HsPCFT migrates as a broad band (~55 kDa), higher than predicted (~50 kDa) in cell lines. Western blot analysis required that membrane preparations not be incubated in the loading buffer above 50 degrees C to avoid aggregation of the protein. Treatment of membrane fractions from HsPCFT-transfected HeLa cells with peptidyl N-glycanase F, or cells with tunicamycin, resulted in conversion to a ~35 kDa species. Substitution of asparagine residues of two canonical glycosylation sites to glutamine, individually, yielded a ~47 kDa protein; substitution of both sites gave a smaller (~35 kDa) protein. Single mutants retained full transport activity; the double mutant retained a majority of activity. Transport function and molecular size were unchanged when the double mutant was hemagglutinin (HA) tagged at either the NH(2) or COOH terminus and probed with an anti-HA antibody excluding degradation of the deglycosylated protein. Wild-type or deglycosylated HsPCFT HA, tagged at amino or carboxyl termini, could only be visualized on the plasma membrane when...Continue Reading

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Citations

Dec 20, 2011·The Journal of Biological Chemistry·Zhanjun HouLarry H Matherly
Dec 17, 2008·The Journal of Biological Chemistry·Rongbao ZhaoI David Goldman
May 1, 2009·American Journal of Physiology. Cell Physiology·Ersin Selcuk UnalI David Goldman
Nov 10, 2013·PloS One·Phaneendra Kumar DuddempudiMichaela Jansen
Feb 12, 2014·Annual Review of Physiology·Michele VisentinI David Goldman
May 17, 2011·Annual Review of Nutrition·Rongbao ZhaoI David Goldman
Mar 20, 2013·Molecular Aspects of Medicine·Rongbao Zhao, I David Goldman
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Apr 6, 2016·FEBS Open Bio·Swapneeta S DateMichaela Jansen
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Jun 24, 2017·American Journal of Physiology. Cell Physiology·Veedamali S SubramanianHamid M Said
Jun 3, 2016·American Journal of Physiology. Cell Physiology·Mitra NajmiI David Goldman
Aug 2, 2017·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·Brijesh K Garg, Ralph H Loring
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Dec 7, 2019·BMC Research Notes·Raquel Caminha DantasKaio Cesar Simiano Tavares
Jul 31, 2012·American Journal of Physiology. Cell Physiology·Daniel Sanghoon ShinDavid I Goldman
Oct 27, 2015·Plant Reproduction·Susanne UeblerThomas Dresselhaus
Sep 22, 2017·Evidence-based Complementary and Alternative Medicine : ECAM·Alexia DickeyKevin Yueju Wang

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Methods Mentioned

BETA
glycosylation
PCR
transfection
protein assay

Software Mentioned

GraphPad
TopPred
HMMTOP
GraphPad PRISM
PredictProtein
TMpred

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