Identification of dynein light chain road block-1 as a novel interaction partner with the human reduced folate carrier.
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 < 0.05) increase in folate uptake. On the other hand, inhibiting the endogenous DYNLRB1 with gene-specific small interfering RNA or pharmacologically with a specific inhibitor (vanadate) led to a significant (P < 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.
Ashokkumar
Balasubramaniem
B
Department of Medical Research, Veterans Affairs Medical Center, Long Beach, California 90822, USA.
Nabokina
Svetlana M
SM
Ma
Thomas Y
TY
Said
Hamid M
HM
D001692:Biological Transport; D018938:Caco-2 Cells; D056445:Cytoplasmic Dyneins; D004398:Dyneins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005492:Folic Acid / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D007413:Intestinal Mucosa / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D058978:Reduced Folate Carrier Protein; D014162:Transfection; D020798:Two-Hybrid System Techniques; D014638:Vanadates / Q000494:pharmacology
C504797:DYNLRB1 protein, human; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; D014638:Vanadates; D005492:Folic Acid; D056445:Cytoplasmic Dyneins; D004398:Dyneins