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Apical membrane targeting and trafficking of the human proton-coupled transporter in polarized epithelia. Am J Physiol Cell Physiol 2008 Jan;294(1):C233-40

Date

11/16/2007

Pubmed ID

18003745

DOI

10.1152/ajpcell.00468.2007

Scopus ID

2-s2.0-38349097299   36 Citations

Abstract

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.

Author List

Subramanian VS, Marchant JS, Said HM

Author

Jonathan S. Marchant PhD Chair, Professor in the Cell Biology, Neurobiology and Anatomy department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Animals
Caco-2 Cells
Cell Line
Cell Membrane
Dogs
Dynactin Complex
Endoplasmic Reticulum
Epithelial Cells
Folic Acid
Humans
Intestinal Mucosa
Kidney
Membrane Transport Proteins
Microscopy, Confocal
Microtubule-Associated Proteins
Microtubules
Mutation
Protein Conformation
Protein Folding
Protein Structure, Tertiary
Protein Transport
Proton-Coupled Folate Transporter
Recombinant Fusion Proteins
Reduced Folate Carrier Protein
Structure-Activity Relationship
Time Factors
Transfection
jenkins-FCD Prod-482 91ad8a360b6da540234915ea01ff80e38bfdb40a