title: Association of TM4SF4 with the human thiamine transporter-2 in intestinal epithelial cells.
abstract: BACKGROUND
  The human thiamine transporter-2 (hTHTR-2) is involved in the intestinal absorption of thiamine. Recent studies with membrane transporters of other nutrients/substrates have shown that they have associated proteins that affect different aspects of their physiology and cell biology. Nothing is known about protein(s) that interact with hTHTR-2 in intestinal epithelial cells and influence its physiological function and/or its cell biology.
  
  
  OBJECTIVE
  The aim of this study was to identify protein partner(s) that interact with hTHTR-2 in human intestinal cells and determine the physiological/biological consequence of that interaction.
  
  
  METHODS
  The yeast split-ubiquitin two-hybrid approach was used to screen a human intestinal cDNA library. GST-pull-down and cellular co-localization approaches were used to confirm the interaction between hTHTR-2 and the associated protein(s). The effect of such an interaction on hTHTR-2 function was examined by (3)H-thiamine uptake assays.
  
  
  RESULTS
  Our screening results identified the human TransMembrane 4 SuperFamily 4 (TM4SF4) as a potential interactor with hTHTR-2. This interaction was confirmed by an in vitro GST-pull-down assay, and by live-cell confocal imaging of HuTu-80 cells co-expressing hTHTR-2-GFP and mCherry-TM4SF4 (the latter displayed a significant overlap of these two proteins in intracellular vesicles and at the cell membrane). Co-expression of hTHTR-2 with TM4SF4 in HuTu-80 cells led to a significant induction in thiamine uptake. In contrast, silencing TM4SF4 with gene-specific siRNA led to a significant decrease in thiamine uptake.
  
  
  CONCLUSIONS
  These results show for the first time that the accessory protein TM4SF4 interacts with hTHTR-2 and influences the physiological function of the thiamine transporter.
authors.affiliation: Departments of Medicine, Physiology and Biophysics, University of California, Irvine, CA, 92697, USA.
authors.forename: Veedamali S
authors.initials: VS
authors.lastname: Subramanian
authors.forename: Svetlana M
authors.initials: SM
authors.lastname: Nabokina
authors.forename: Hamid M
authors.initials: HM
authors.lastname: Said
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D004386:Duodenum / Q000166:cytology / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D060888:Real-Time Polymerase Chain Reaction; D013831:Thiamine / Q000378:metabolism*
chemical_list: D015415:Biomarkers; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; C492291:SLC19A3 protein, human; C429031:TM4SF4 protein, human; D013831:Thiamine