Identification of dynein light chain road block-1 as a novel interaction partner with the human reduced folate carrier

Abstract

Ashokkumar B, Nabokina SM, Ma TY, Said HM. Identification of dynein light chain road block-1 as a novel interaction partner with the human reduced folate carrier. Am J Physiol Gastrointest Liver Physiol 297: G480 -G487, 2009. First published July 1, 2009 doi:10.1152/ajpgi.00154.2009.-The reduced folate carrier (RFC) is a major folate transport system in mammalian cells. RFC is highly expressed in the intestine and believed to play a role in folate absorption. Studies from our laboratory and others have characterized different aspects of the intestinal folate absorption process, but little is known about possible existence of accessory protein(s) that interacts with RFC and influences its physiology and/or cell biology. We investigated this issue by employing a bacterial two-hybrid system to screen a BacterioMatch II human intestinal cDNA library using the large intracellular loop between transmembrane domains 6 and 7 of the human RFC (hRFC) as bait. Our screening has resulted in the identification of dynein light chain road block-1 (DYNLRB1) as an interacting partner with hRFC. Existence of a direct protein-protein interaction between hRFC and DYNLRB1 was confirmed by in vitro pull-down assay and in vivo mammalian two-hybrid luciferase assay and coimmunoprecipitation analysis. Furthermore, confocal imaging of live human intestinal epithelial HuTu-80 cells demonstrated colocalization of DYNLRB1 with hRFC. Coexpression of DYNLRB1 with hRFC led to a significant (P Ͻ 0.05) increase in folate uptake. On the other hand, inhibiting the endogenous DYNLRB1 with genespecific small interfering RNA or pharmacologically with a specific inhibitor (vanadate) led to a significant (P Ͻ 0.05) decrease in folate uptake. This study demonstrates for the first time the identification of DYNLRB1 as an interacting protein partner with hRFC. Furthermore, DYNLRB1 appears to influence the function and cell biology of hRFC. folate transport; protein-protein interactions; two-hybrid analysis; accessory proteins

Structure-function activity of the human sodium-dependent multivitamin transporter: role of His115 and His254

“…Cell surface proteins were biotinylated as described by us and others before (1,32). Briefly, after 48 h of transfection ARPE19 cells were incubated with sulfo-NHS-SSbiotin (Pierce Biotechnology, Rockford, IL) in biotinylation buffer (PBS, pH 7.5, supplemented with 10 mM triethanolamine, 2 mM CaCl2, and 150 mM NaCl).…”

Association of PDZ-containing protein PDZD11 with the human sodium-dependent multivitamin transporter

“…To generate pGEX-4T-1-PDZD11, the open reading frame of human PDZD11 (OriGene, Rockville, MD) was inserted in frame into BamHI/ XhoI sites of pGEX-4T-1 vector (Invitrogen, Carlsbad, CA). GST-PDZD11 fusion protein and GST were produced in BL-21 Escherichia coli cells and used in GST pull-down assay as described before (1). Briefly, cleared (14,000 g) postnuclear extract of Caco-2 cells (1 mg of total soluble protein) was incubated with either GST-PDZD11 (100 g) or GST (100 g) bound to glutathione-Sepharose4B beads (GE Healthcare, Piscataway, NJ) in CelLytic M cell lysis reagent (Sigma, St. Louis, MO) for 2 h at 4°C.…”

Molecular determinants dictating cell surface expression of the human sodium-dependent vitamin C transporter-2 in human liver cells

“…The motor proteins kinesin, dynein, and myosin play important roles in delivering proteins, including vitamin transporters, to the cell surface in many diverse cell types (1,3,8,12,29,33). However, the role of these molecular motors in regulating hSVCT2 functional cell surface expression in HepG2 cells, or any other cell type, is unclear.…”