Subcellular localization of the transmembrane inner ear (Tmie) protein in a stable Tmie-expressing cell line

Abstract

Mutations in the transmembrane inner ear (Tmie) gene, which encodes the Tmie protein, have been attributed to deafness autosomal recessive 6 (DFNB6), an autosomal nonsyndromic recessive hearing loss disorder. Although the Tmie gene was identified a few years ago, little is known about subcellular localization of the Tmie protein. In order to address this, we developed a stable cell line expressing Tmie protein. The expression of Myc-tagged Tmie protein was confirmed by Western blot analysis using an anti-Myc antibody and localization of the Tmie protein was confirmed by immunostaining, using the anti-Myc antibody as well as the anti-tmie antibody. Our study demonstrates that the Tmie protein is localized mostly in the cellular membrane and to a lesser extent in cytoplasm. These results suggest that our Tmie expressing stable cell line provides a suitable in vitro model to explore Tmie synthesis and functions.

Keywords: HEK293; Transmembrane inner ear protein; hearing loss disorder; subcellular localization.

Figure 1

The map and sequences of the expressing vector containing the Tmie gene (pcDNA 3.1-Myc-His).

Figure 2

Tmie expression in Tmie-HEK293 cell lines. Tmie (~21 kDa) expression was detected by western blotting using an anti-Myc antibody. Lane 1: lysate from HEK293 cells transfected expressing an empty vector, Lane 2: lysate from HEK293 cells transfected expressing a vector containing the Tmie gene.

Figure 3

Subcellular localization of Tmie in Tmie-expressing cells (Tmie-HEK293). Expression of the Tmie protein was detected in the stable cell line by confocal microscopy. Tmie protein expression was found in the cellular membrane (red) and to a lesser extent in cytoplasm. Upper panels are expressing vector-treated cells. Lower panels are mock-treated cells. Merge indicates merged images of Myc (green) and Tmie (red) immunofluorescence. (Scale bar=20 µm).