Premium
Effect of clinical mutations on functionality of the hRFT‐2 in intestinal epithelial cells
Author(s) -
Nabokina Svetlana,
Subramanian Veedamali S,
Said Hamid M
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.885.24
Subject(s) - mutant , transfection , biotinylation , microbiology and biotechnology , transporter , green fluorescent protein , chemistry , translation (biology) , messenger rna , gene , biology , biochemistry
The Brown‐Vialetto‐Van Laere syndrome (BVVLS) is a rare neurological disorder believed to be due to mutations in the C20orf54 gene, which encodes the human riboflavin (RF) transporter −2 (hRFT‐2). However, there is nothing known about the effect of these mutations on functionality of hRFT‐2, a protein that is expressed in a variety of tissues with high expression in the intestine. We addressed this issue for a number of the mutants using the Caco‐2 cells. Our results showed significant inhibition in RF uptake in Caco‐2 cells expressing W17R, P28T, E36K, E71K, and R132W hRFT‐2 mutants. The inhibition in RF transport in these mutants were not due to a decrease in transcription and/or translation of the hRFT‐2, since mRNA and protein levels of the carrier were similar in mutants and wild‐type hRFT‐2. Confocal images of live Caco‐2 cells transfected with hRFT‐2 mutants (fused with GFP) showed the P28T, E36K, E71K, and R132W mutants were retained within the ER, while the W17R mutant was expressed at the cell membrane; the latter was further confirmed by cell surface biotinylation assay. These results show for the first time that BVVLS associated mutations in hRFT‐2 affect the transporter functionality and that this effect is mediated via alterations in membrane targeting and/or activity of the transporter. [Supported by grants from the DVA and NIH (DK 58057 and DK 84094)].