Expression and characterization of transmembrane and coiled-coil domain family 3

Abstract

Transmembrane and coiled-coil domain family 3 (TMCC3) has been reported to be expressed in the human brain; however, its function is still unknown. Here, we found that expression of TMCC3 is higher in human whole brain, testis and spinal cord compared to other human tissues. TMCC3 was expressed in mouse developing hind brain, lung, kidney and somites, with strongest expression in the mesenchyme of developing tongue. By expression of recombinant TMCC3 and its deletion mutants, we found that TMCC3 proteins self-assemble to oligomerize. Immunostaining and confocal microscopy data revealed that TMCC3 proteins are localized in endoplasmic reticulum through transmembrane domains. Based on immunoprecipitation and mass spectroscopy data, TMCC3 proteins associate with TMCC3 and 14-3-3 proteins. This supports the idea that TMCC3 proteins form oligomers and that 14-3-3 may be involved in the function of TMCC3. Taken together, these results may be useful for better understanding of uncharacterized function of TMCC3. [BMB Reports 2016; 49(11): 629-634].

Fig. 1

Expression of TMCC3 in human tissues: The human tissue cDNA was analyzed using standard real-time PCR to check TMCC3 expression. The β-actin expression level was used as a control to normalize the amount of template cDNA.

Fig. 2

Expression pattern of TMCC3 in developing embryo: Mouse embryo sections were prepared at embryo day 12 (E12) and at embryo day 14 (E14), and analyzed by in situ hybridization using antisense and sense probes of TMCC3. tm: tongue mesenchyme, hb: hind brain, lu: lung, oe: oral epithelium, tg: trigeminal ganglion, kd: kidney, sm: somites.

Fig. 3

Expression and characterization of TMCC3 and its deletion mutant proteins: (A) Map of full length TMCC3 and deletion mutant expression vectors. (B) Expression of recombinant proteins in HEK 293 cells. Cell lysates were prepared from HEK 293 cells transfected with TMCC3 and its deletion mutant expression vectors. The cell lysates were separated by SDS-PAGE and analyzed using Western blotting with anti-Myc antibody. The amount of β-actin was used as a control. (C) Localization of TMCC3. HEK 293 cells expressing TMCC3 recombinant proteins were visualized by immunofluorescence staining using anti-Myc antibody and confocal microscopy. (D) Differential localization of TMCC3 recombinant proteins in cytosolic and membrane fraction. Protein fractions were resolved by SDS-PAGE and analyzed by Western blotting with anti-Myc antibody.

Fig. 4

Oligomerization of TMCC3 and association of TMCC3 with 14-3-3: Immuno-complexes containing TMCC3 and anti-Myc-tag antibody were separated by 12.5% SDS-PAGE. After staining with Coomassie brilliant blue G-250, the co-immuno-precipitated protein bands were examined by mass spectrometry. The protein bands identified were indicated with arrows.