Cys(294) is essential for the function of the human sodium-dependent multivitamin transporter.
The sodium-dependent multivitamin transporter (SMVT) plays an important role in biotin uptake in the intestine and other cell types. While significant knowledge has been gained with regard to regulation and cell biology of the SMVT system, there is little known about its structure-function relationships. Here we examined the role of each of the ten conserved (among species) cysteine residues in the function of the human SMVT (hSMVT) using site-directed mutagenesis. Our results showed a significant impairment in biotin uptake only in cells transfected with hSMVT mutated at Cys(294), but not at the other conserved cysteine residues; the impairment in biotin uptake caused by mutating Cys(294) was not related to the polar status of substituting amino acid. The inhibition in hSMVT function upon mutating Cys(294) was mediated via a significant reduction in the V(max), but not the apparent K(m), of the biotin uptake process, suggesting a decrease in the number (and/or activity) of hSMVT but not affinity. Biotinylation assay confirmed this suggestion by showing a marked reduction in the level of expression of the mutated protein at the cell membrane, without affecting total cellular level of induced hSMVT. These results show an important role for Cys(294) in the function and cell biology of hSMVT.
Department of Medicine, University of California, Irvine, CA 92697, USA.
Abhisek
A
Ghosal
Hamid M
HM
Said
D001692:Biological Transport; D001710:Biotin / Q000378:metabolism; D019921:Biotinylation; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016133:Polymerase Chain Reaction; D012160:Retina / Q000166:cytology / Q000378:metabolism*; D013329:Structure-Activity Relationship; D027981:Symporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D017931:DNA Primers; D027981:Symporters; C119981:biotin transporter; D001710:Biotin; D003545:Cysteine