Nuclear trafficking of photoreceptor protein Crx: The targeting sequence and pathologic implications

PURPOSE. To identify the targeting sequence controlling the nuclear transpo rt of the photoreceptor-specific transcription factor cone-rod homeobox (Cr x) protein and to address the question of whether disease-causing Crx mutat ions disrupt the nuclear trafficking of the Crx protein. METHODS. A series of cDNA fragments encoding Crx protein with deleted C ter mini were generated from mouse Crx cDNA by polymerase chain reaction (PCR). Point mutations were introduced into Crx coding sequence through PCR-based , site-directed mutagenesis. These mutated Crx fragments and the wild-type Crx were fused to cDNA encoding the jellyfish green fluorescent protein (GF P) and were transiently expressed in human embryonic kidney (HEK) 293T cell s. Twelve to 48 hours after transfection, the living cells were counterstai ned with the red fluorescent nucleic acid dye SYTO 59 and examined with epi fluorescence and confocal microscopy to determine the subcellular localizat ion of Crx fusion proteins. RESULTS. GFP expressed without a fusion partner was distributed evenly thro ughout the cells, whereas the wild-type Crx protein fused to GFP was locali zed only in the nucleus. GFP-tagged Crx proteins truncated at residues 107 or 165, demonstrated exclusive nuclear localization. In contrast, Crx fusio n proteins truncated at residues 88, 79, 44, and 36, were located equally i n both the cytoplasm and the nucleus. These results demonstrate that the nu clear localization signal (NLS) of Crx appears to reside in the amino acids between residue 88 and 107, which is surprising because the putative NLSs identified by prosite search are at residues 36 to 43 and 116 to 122. Furth er, a Crx fusion protein truncated at residue 99 was localized within the n ucleus in the majority of the transfected cells, and two point mutations at residues 88 (K88T) and 98 (R98L) disrupted the nuclear localization, which indicates that the sequence between 88 and 98 in the C terminus of the Crx homeodomain contains a NLS that is essential for targeting Crx to the nucl eus. However, the fusion protein truncated at residue 99 did not produce a complete nuclear localization in every transfected cell, suggesting that th e Gln-rich domain at residues 99 to 106 is also required for the full accum ulation of Crx protein in the nucleus. Two point mutations of Crx, R41W and E80A, that cause cone-rod dystrophy in humans and lie within the homeodoma in but outside the NLS did not disrupt the nuclear localization of Crx prot ein, but a R90W mutation of Crx that causes human Leber congenital amaurosi s (LCA) and resides within the NLS resulted in the fusion protein localized in both nuclei and cytoplasm in majority (51% to 69%) of the transfected c ells. CONCLUSIONS. The wild-type Crx protein is localized within the nucleus. The putative NLSs of Crx at residues SG to 43 and 116 to 122 are not essential . The minimal NLS necessary for the nuclear transport of Crx protein is loc ated at residues 88 to 98 in the C terminus of the homeodomain. The R90W mu tation of Crx found in LCA disrupts the nuclear transport of the mutant pro tein. The defective nuclear trafficking of Crx protein may be a part of the molecular mechanism of this early-onset retinal degeneration.