A pat7 nuclear localization signal targets the protein encoded by NORPEG gene into retinal pigment epithelial (RPE) cell nuclei

Abstract

Abstract: : Purpose: NORPEG (novel retinal pigment epithelial cell gene) is a retinoic acid regulated gene that we originally characterized from the human retinal pigment epithelial cell line ARPE–19 (Kutty et al., J. Biol. Chem. 276:2831, 2001). It encodes a protein consisting of 980 amino acid residues containing an ankyrin repeat region and a coiled–coil domain, and is developmentally regulated. Analysis of its amino acid sequence revealed the presence of a pat7 type nuclear localization signal (PKTRKAP) starting at amino acid residue 270. The purpose of this study was to investigate whether this potential nuclear localization signal can target the NORPEG protein into cell nuclei. Methods: Part of the open reading frame of NORPEG encoding the N–terminal 286 amino acid residues, containing the pat7 nuclear localization signal, was cloned into the phrGFP–N1 mammalian expression vector. ARPE–19 and COS–7 cells were transfected with this construct and the expression of the green fluorescent protein (GFP)–fusion protein was examined with a confocal microscope. Cell nuclei were stained using DAPI. Cells were also analyzed after immunostaining for nucleolin, a marker for cell nuclei. Results:Transfected ARPE–19 and COS–7 cells expressing GFP fused to N–terminal 286 amino acid residues of the NORPEG showed nuclear–specific fluorescence. The nuclear origin of GFP fluorescence was confirmed by DAPI staining of the cell nuclei. GFP fluorescence was concentrated in nucleoli, based on co–localization studies with antibodies to nucleolin. Conclusions: The pat7 nuclear localization signal present in the NORPEG protein is able to target a GFP–fusion protein to the cell nucleus. The potential nuclear localization of the protein encoded by NORPEG, a gene regulated during development and by retinoic acid, suggests that this protein may play an important role in RPE cell structure and function.

Keywords: protein structure/function • retinal pigment epithelium • immunohistochemistry