title: Membrane targeting and intracellular trafficking of the human sodium-dependent multivitamin transporter in polarized epithelial cells.
abstract: The human sodium-dependent multivitamin transporter (hSMVT) mediates sodium-dependent uptake of biotin in renal and intestinal epithelia. To date, however, there is nothing known about the structure-function relationship or targeting sequences in the hSMVT polypeptide that control its polarized expression within epithelia. Here, we focused on the role of the COOH-terminal tail of hSMVT in the targeting and functionality of this transporter. A full-length hSMVT-green fluorescent protein (GFP) fusion protein was functional and expressed at the apical membrane in renal and intestinal cell lines. Microtubule disrupting agents disrupted the mobility of trafficking vesicles and impaired cell surface delivery of hSMVT, which was also prevented in cells treated with dynamitin (p50), brefeldin, or monensin. Progressive truncation of the COOH-terminal tail impaired the functionality and targeting of the transporter. First, biotin transport decreased by approximately 20-30% on deletion of up to 15 COOH-terminal amino acids of hSMVT, a decrease mimicked solely by deletion of the terminal PDZ motif (TSL). Second, deletions into the COOH-terminal tail (between residues 584-612, containing a region of predicted high surface accessibility) resulted in a further drop in hSMVT transport (to approximately 40% of wild-type). Third, apical targeting was lost on deletion of a helical-prone region between amino acids 570-584. We conclude that the COOH tail of hSMVT contains several determinants important for polarized targeting and biotin transport.
authors.affiliation: Department of Medicine, University of California, Irvine, USA.
authors.forename: Veedamali S
authors.initials: VS
authors.lastname: Subramanian
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Marchant
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Boulware
authors.forename: Thomas Y
authors.initials: TY
authors.lastname: Ma
authors.forename: Hamid M
authors.initials: HM
authors.lastname: Said
mesh_terms: D000818:Animals; D001710:Biotin / Q000378:metabolism*; D020126:Brefeldin A / Q000494:pharmacology; D018938:Caco-2 Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D016764:Cell Polarity; D003593:Cytoplasm / Q000378:metabolism; D004285:Dogs; D000072159:Dynactin Complex; D004398:Dyneins / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism*; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism; D008985:Monensin / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D027981:Symporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D022161:Transport Vesicles / Q000378:metabolism; D050257:Tubulin Modulators / Q000494:pharmacology
chemical_list: C000606361:DCTN2 protein, human; D000072159:Dynactin Complex; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; D027981:Symporters; D050257:Tubulin Modulators; C119981:biotin transporter; D049452:Green Fluorescent Proteins; D020126:Brefeldin A; D001710:Biotin; D008985:Monensin; D004398:Dyneins