Identification of transmembrane protein 237 as a novel interactor with the intestinal riboflavin transporter-3 (RFVT-3): role in functionality and cell biology

Abstract

The apically localized riboflavin (RF) transporter-3 (RFVT-3) is involved in intestinal absorption of vitamin B2. Previous studies have characterized different physiological/biological aspects of the RFVT-3, but there is a lack of knowledge regarding possible existence of interacting partner(s) and consequence of interaction(s) on its function/cell biology. To address the latter, we performed yeast two-hybrid (Y2H) screening of a human colonic cDNA library and have identified transmembrane protein 237 (TMEM237) as a putative interactor with the human (h)RFVT-3; the interaction was further confirmed via "1-by-1" Y2H assay that involved appropriate positive and negative controls. TMEM237 was found to be highly expressed in human native intestine and in human intestinal epithelial cell lines; further, confocal images showed colocalization of the protein with hRFVT-3. The interaction between TMEM237 with hRFVT-3 in human intestinal epithelial HuTu-80 cells was established by coimmunoprecipitation. Expressing TMEM237 in HuTu-80 cells led to a significant induction in RF uptake, while its knockdown (with the use of gene-specific siRNA) led to a significant reduction in uptake. Transfecting TMEM237 into HuTu-80 cells also led to a marked enhancement in hRFVT-3 protein stability (reflected by an increase in the protein half-life). Interestingly, the level of expression of TMEM237 was found to be markedly reduced following treatment with TNF-α (a proinflammatory cytokine that inhibits intestinal RF uptake), while its expression was significantly upregulated following treatment with butyrate (an inducer of intestinal RF uptake). These findings identify TMEM237 as an interactor with the intestinal hRFVT-3 and show that the interaction has physiological/biological significance.

Keywords: RFVT-3; TMEM237; interacting partner; intestine; riboflavin transporter.

Fig. 1.

Direct confirmation of interaction between the human riboflavin transporter-3 (hRFVT-3) and its putative interactor TMEM237 using “1-by-1” yeast two-hybrid (Y2H) interaction assay. A: schematic representation of the full-length hRFVT-3 (bait) and TMEM237 (prey) with the coding sequence 242–469 amino acid (aa) of hRFVT-3 and 35–255 aa of TMEM237 used for the “1-by-1” Y2H studies boxed. B: yeast cells were grown in selective medium DO-2 and DO-3. The DO-2 selective medium (lacking tryptophan and leucine) was used as a growth control and to verify the presence of the bait and prey vectors whereas the DO-3 selective medium (without tryptophan, leucine, and histidine) was selected for the interaction between bait and prey. (B1) Commercial vendor (Hybergenics) positive control [interaction between the SMAD (as bait) and SMURF (as prey)]; (B2) negative control [hRFVT-3 (bait) and pP6φ empty (prey) vector]; (B3) negative control (empty bait vector pB66φ and TMEM237); (B4) positive interaction between hRFVT-3 and TMEM237.

Fig. 2.

A and B: expression of TMEM237 in human native small and large intestine (A) and in different human-derived intestinal epithelial cell lines (B). Human small and large intestinal cDNA samples that were pooled from 5 to 12 individuals (cat. no. 636746; Clontech), as well as cDNA samples prepared in our laboratory from different human-derived intestinal epithelial cell lines were used. Quantitative RT-PCR was performed using TMEM237 and β-actin primers. C: subcellular colocalization of GFP-human riboflavin transporter-3 (hRFVT-3) and TMEM237-mCherry in HuTu-80 cells. Cells were transiently cotransfected with GFP-hRFVT-3 and TMEM237-mCherry plasmid constructs. After 48 h of transfection, live confocal imaging was performed as described in materials and methods. Fluorescence of GFP (left), mCherry (middle), and an overlay (right) are shown for coexpressing cell.

Fig. 3.

Coimmunoprecipitation (co-IP) of TMEM237 with human riboflavin transporter-3 (hRFVT-3) in HuTu-80 cells. Cells that were cotransiently transfected with the TMEM237- mCherry and GFP-hRFVT-3 were lysed in RIPA buffer and then subjected to co-IP using anti-hRFVT-3 antibodies (Ab°) (see materials and methods). co-IP complex was separated in NuPAGE 4–12% Bis-Tris minigel and analyzed by Western blotting (WB) using the anti-TMEM237 antibodies (Ab°). Control, co-IP complex was obtained from mock-transfected HuTu-80 cells. Coimmunoprecipitation studies were repeated on 3 separate occasions using 3 different cell lysates.

Fig. 4.

Overexpression of TMEM237 increases [³H]riboflavin (RF) uptake by HuTu-80 cells that stably express human riboflavin transporter-3 (hRFVT-3). Cells stably expressing the GFP-hRFVT-3 were transiently transfected with TMEM237-mCherry. Forty-eight hours following transfection, carrier-mediated [³H]-RF uptake (14 nM; pH 7.4; 5 min) was determined. TMEM237 coexpressed with hRFVT-3 caused significant (**P < 0.01) increase in [³H]-RF uptake compared with hRFVT-3 expression alone. Data are means ± SE of at least 4 separate experiments.

Fig. 5.

Effect of knocking down TMEM237 using gene-specific siRNA on carrier-mediated [³H]riboflavin (RF) uptake by HuTu-80 cells. Cells were treated with the TMEM237-specific siRNAs or with scrambled (nontargeting negative) siRNA (control) as described in materials and methods. A: initial rate (5 min) of carrier-mediated [³H]-RF uptake (14 nM) was determined (see materials and methods) 48 h following transfection with siRNA. B: Western blot was performed using 60 μg total protein from HuTu-80 cells that were transfected with the TMEM237 specific siRNA or with scrambled siRNA (control). The blot was probed with the polyclonal antibodies directed against TMEM237 and data were normalized relative to β-actin. C: quantitative RT-PCR was performed using total RNA isolated from HuTu-80 cells and gene-specific primers as described in materials and methods. Data are means ± SE of at least 4 separate sets of determinations (*P < 0.05; **P < 0.01).

Fig. 6.

Effect of TMEM237 on stability of the human riboflavin transporter-3 (hRFVT-3) and on uptake of [³H]-RF by HuTu-80 cells. A: cells stably expressing the GFP-hRFVT-3 were transfected with TMEM237-mCherry and then (48 h later) treated with 100 μg/ml of cycloheximide (for 3 h). Cells were then lysed, the lysates were analyzed by Western blotting using anti-hRFVT-3 antibodies, and the specific protein bands were then visualized/quantified by LI-COR imaging system. Ai and Aii: for normalization, the same blot was probed with the anti-β-actin antibody. hRFVT-3-GFP (Ai) and endogenous hRFVT-3 (Aii). B: carrier-mediated [³H]-RF uptake (14 nM; pH 7.4; 5 min) was determined as described in materials and methods. All data are means ± SE of at least 4 separate determinations (**P < 0.01).

Fig. 7.

A and B: effect of proinflammatory cytokine TNF-α (A) and butyrate (B) on level of expression of TMEM237 protein and mRNA in human intestinal Caco-2 cells. A: cells were treated with TNF-α (20 ng/ml for 48 h) followed by determination of TMEM237 protein (Western blotting; Ai) and mRNA (quantitative RT-PCR; Aii) level. B: cells were treated with butyrate (1 mM; 24 h) followed by determination of TMEM237 protein (Western blotting; Bi) and mRNA (quantitative RT-PCR; Bii) level. Normalization was done relative to β-actin. Data are means ± SE of at least 4–5 different sets of independent experiments. **P < 0.01.