Apical membrane targeting and trafficking of the human proton-coupled transporter in polarized epithelia.
The human proton-coupled folate transporter (hPCFT) is a recently discovered intestinal transporter involved in folate uptake in epithelia (and possibly other cells). Little is currently known about the structure-function relationship of the different domains of this transporter, particularly which regions are important for substrate transport as well as targeting of the transporter to the apical cell surface of polarized cells. Here we have investigated the role of the COOH-terminal domain and a well-conserved sequence separating transmembrane (TM) domains TM2 and TM3 (DXXGRR; amino acids 109-114) speculated by others to be important for transport function. Using live cell imaging approaches, we show that 1) an hPCFT-yellow fluorescent protein construct is functionally expressed at the apical membrane domain and is localized differentially to the human reduced folate carrier; 2) the predicted cytoplasmic COOH-terminal region of hPCFT is not essential for apical targeting or transporter functionality; 3) mutations that ablate a consensus beta-turn sequence separating predicted TM2 and TM3 abolished apical [(3)H]folic acid uptake as a consequence of endoplasmic reticulum retention of mutant, likely misfolded, transporters; and 4) cell surface delivery of hPCFT is disrupted by microtubule depolymerization or by overexpression of the dynactin complex dynamitin (p50). For the first time, our data present information regarding structure-function and membrane targeting of the hPCFT polypeptide, as well as the mechanisms that control its steady-state expression in polarized cells.
Subramanian
Veedamali S
VS
Departments of Medicine, University of California, Irvine, California, USA.
Marchant
Jonathan S
JS
Said
Hamid M
HM
D000818:Animals; D018938:Caco-2 Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D004285:Dogs; D000072159:Dynactin Complex; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism*; D005492:Folic Acid / Q000378:metabolism*; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism* / Q000473:pathology; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D058979:Proton-Coupled Folate Transporter; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D058978:Reduced Folate Carrier Protein; D013329:Structure-Activity Relationship; D013997:Time Factors; D014162:Transfection
C000606361:DCTN2 protein, human; D000072159:Dynactin Complex; D026901:Membrane Transport Proteins; D008869:Microtubule-Associated Proteins; D058979:Proton-Coupled Folate Transporter; D011993:Recombinant Fusion Proteins; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; C505058:SLC46A1 protein, human; D005492:Folic Acid