Cellular Localization of 1363, an Uncharacterized Gene Identified in a Screen for Differentially Expressed Light–Induced Retinal Degeneration Genes in the Rat

Abstract

Purpose: : Light–induced retinal degeneration (LIRD) progresses by an oxidative stress mediated active cell death process. In an effort to identify genes that define a retinal degeneration molecular phenotype we screened a rat retinal cDNA library for genes that are differentially expressed during LIRD. One previously uncharacterized gene, 1363, isolated from this screen was chosen for further study. Previous work on 1363 identified two nuclear localization signals (NLS). In an effort to further elucidate the function of this gene we carried out studies to determine the cellular localization of this protein.

Methods: : The coding sequence of 1363 was inserted into a pEGFP–C1 vector to create the wild–type construct. For the mutant construct the nuclear localization signals were mutated by way of site–directed mutagenesis. COS–7 cells were transfected with either wild–type or NLS mutant GFP–1363 fused constructs. Transfected cells were also treated with chemical agents including colcemid, hydrogen peroxide, paraquat, and staurosporine to observe cellular localization under various stress conditions. Cellular localization was analyzed by fluorescence microscopy.

Results: : Cells transfected with the GFP–1363 wild–type construct displayed a filamentous pattern in the cytosol, which resembles the pattern of Alpha–tubulin distribution. In contrast to this, the mutant constructs displayed a punctate cytoplasmic localization. When the cells were treated with colcemid, which disrupts microtubules, the filamentous pattern seen with the wild–type construct was disrupted suggesting an association between 1363 and microtubules. Treatment with hydrogen peroxide, paraquat, and staurosporine resulted in some wild–type transfected cells displaying nuclear or perinuclear localization.

Conclusions: : Despite the presence of the two NLS in the protein, the protein showed predominant cytosolic localization under normal conditions. Mutations in the NLS resulted in an aberrant cellular distribution suggesting that the NLS sequence may be relevant to its normal cytosolic localization. Exposure to oxidative stress/apoptosis inducing chemicals resulted in altered localization of the wild–type protein suggesting that this relocalization is stress responsive.

Keywords: retinal degenerations: cell biology • oxidation/oxidative or free radical damage • gene/expression