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Biochim Biophys Acta. 2011 Apr;1808(4):1140-5. doi: 10.1016/j.bbamem.2011.01.008. Epub 2011 Jan 20.

Vulnerability of the cysteine-less proton-coupled folate transporter (PCFT-SLC46A1) to mutational stress associated with the substituted cysteine accessibility method.

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1
Department of Medicine and Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY, USA.

Abstract

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.

PMID:
21256110
PMCID:
PMC3062655
DOI:
10.1016/j.bbamem.2011.01.008
[Indexed for MEDLINE]
Free PMC Article
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