Vulnerability of the cysteine-less proton-coupled folate transporter (PCFT-SLC46A1) to mutational stress associated with the substituted cysteine accessibility method.
The proton-coupled transporter (PCFT) mediates intestinal folate absorption and folate transport from blood across the choroid plexus. The membrane topology of PCFT has been defined using the substituted cysteine accessibility method; an intramolecular disulfide bond between the Cys 66 and 298 residues, in the first and fourth extracellular loops, respectively, is present but not essential for function. The current report describes Lys 422 mutations (K422C, K422E) that have no effect on transport activity when introduced into wild-type PCFT but result in a marked loss of activity when introduced into a Cys-less PCFT which is otherwise near-fully functional. The loss of activity of both mutant PCFTs was shown to be due to impaired protein stability and expression. Additional studies were conducted with the K422C mutation in Cys-less PCFT. The impact of re-introduction of one, two, three or five, Cys residues was assessed. While there were some differences in the impact of the different Cys residues re-introduced, restoration was attributed more to a cumulative effect rather than the specific role of individual Cys residues. Preservation of the Cys66-Cys298 intramolecular disulfide bond was not required for stability of the K422C protein. These observations are relevant to studies with Cys-less transporters utilized for the characterization of proteins with the substituted cysteine accessibility method and indicate that functional defects detected in a Cys-less protein, when the tertiary structure of the molecule is stressed, are not necessarily relevant to the wild-type protein.
Department of Medicine and Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY, USA.
Rongbao
R
Zhao
Daniel Sanghoon
DS
Shin
I David
ID
Goldman
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001692:Biological Transport / Q000235:genetics; D015153:Blotting, Western; D003545:Cysteine / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D055550:Protein Stability; D058979:Proton-Coupled Folate Transporter / Q000235:genetics* / Q000378:metabolism
D058979:Proton-Coupled Folate Transporter; D008239:Lysine; D003545:Cysteine
Am J Physiol Cell Physiol. 2010 Nov;299(5):C1153-61
20686069
Mol Genet Metab. 2010 Mar;99(3):325-8
20005757
Blood. 2010 Dec 9;116(24):5162-9
20805364
Clin Cancer Res. 2004 Jan 15;10(2):718-27
14760095
Annu Rev Biophys Biomol Struct. 2006;35:67-91
16689628
Medicine (Baltimore). 2002 Jan;81(1):51-68
11807405
Curr Opin Investig Drugs. 2010 Dec;11(12):1409-23
21154123
Mol Pharmacol. 2008 Sep;74(3):854-62
18524888
Clin Cancer Res. 2004 Dec 15;10(24):8735-42
15623659
Biochim Biophys Acta. 2008 Jun;1778(6):1407-14
18405659
J Pediatr. 2008 Sep;153(3):435-7
18718264
Am J Physiol Cell Physiol. 2009 Jul;297(1):C66-74
19403800
Mol Pharmacol. 2010 Oct;78(4):577-87
20601456
J Biol Chem. 2009 Jun 26;284(26):17846-57
19389703
Am J Physiol Cell Physiol. 2007 Nov;293(5):C1669-78
17898134
Blood. 2007 Aug 15;110(4):1147-52
17446347
Biochem Biophys Res Commun. 2009 Aug 28;386(3):426-31
19508863
J Biol Chem. 2009 Aug 14;284(33):22123-22132
19549785
Clin Immunol. 2009 Dec;133(3):287-94
19740703
Mol Cancer Ther. 2007 Feb;6(2):404-17
17308042
Blood. 2008 Sep 1;112(5):2055-61
18559978
Biochemistry. 2010 Apr 6;49(13):2925-31
20225891
Mol Cancer Ther. 2009 Aug;8(8):2424-31
19671745
Methods Enzymol. 1998;293:123-45
9711606
Biochemistry. 2002 May 21;41(20):6346-53
12009896
Cell. 2006 Dec 1;127(5):917-28
17129779