| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
From the Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, Connecticut.
PURPOSE. To identify the targeting sequence controlling the nuclear transport of the photoreceptor-specific transcription factor cone–rod homeobox (Crx) protein and to address the question of whether disease-causing Crx mutations disrupt the nuclear trafficking of the Crx protein.
METHODS. A series of cDNA fragments encoding Crx protein with deleted C termini 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 (GFP) and were transiently expressed in human embryonic kidney (HEK) 293T cells. Twelve to 48 hours after transfection, the living cells were counterstained with the red fluorescent nucleic acid dye SYTO 59 and examined with epifluorescence and confocal microscopy to determine the subcellular localization of Crx fusion proteins.
RESULTS. GFP expressed without a fusion partner was distributed evenly throughout the cells, whereas the wild-type Crx protein fused to GFP was localized only in the nucleus. GFP-tagged Crx proteins truncated at residues 107 or 165, demonstrated exclusive nuclear localization. In contrast, Crx fusion proteins truncated at residues 88, 79, 44, and 36, were located equally in both the cytoplasm and the nucleus. These results demonstrate that the nuclear 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. Further, a Crx fusion protein truncated at residue 99 was localized within the nucleus 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 nucleus. However, the fusion protein truncated at residue 99 did not produce a complete nuclear localization in every transfected cell, suggesting that the Gln-rich domain at residues 99 to 106 is also required for the full accumulation 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 homeodomain but outside the NLS did not disrupt the nuclear localization of Crx protein, but a R90W mutation of Crx that causes human Leber congenital amaurosis (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 cells.
CONCLUSIONS. The wild-type Crx protein is localized within the nucleus. The putative NLSs of Crx at residues 36 to 43 and 116 to 122 are not essential. The minimal NLS necessary for the nuclear transport of Crx protein is located at residues 88 to 98 in the C terminus of the homeodomain. The R90W mutation of Crx found in LCA disrupts the nuclear transport of the mutant protein. The defective nuclear trafficking of Crx protein may be a part of the molecular mechanism of this early-onset retinal degeneration.
This article has been cited by other articles:
|
|
 |
|
  T. Tao, J. Lan, G. L. Lukacs, R. J. G. Hache, and F. Kaplan Importin 13 Regulates Nuclear Import of the Glucocorticoid Receptor in Airway Epithelial Cells Am. J. Respir. Cell Mol. Biol., December 1, 2006; 35(6): 668 - 680. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Liu, J. Qiu, F. Cheng, Y. Chu, Y. Yoto, M. G. O'Sullivan, K. E. Brown, and D. J. Pintel Comparison of the Transcription Profile of Simian Parvovirus with That of the Human Erythrovirus B19 Reveals a Number of Unique Features J. Virol., December 1, 2004; 78(23): 12929 - 12939. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. L. Whitaker and B. E. Knox Conserved Transcriptional Activators of the Xenopus Rhodopsin Gene J. Biol. Chem., November 19, 2004; 279(47): 49010 - 49018. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. E. Ploski, M. K. Shamsher, and A. Radu Paired-Type Homeodomain Transcription Factors Are Imported into the Nucleus by Karyopherin 13 Mol. Cell. Biol., June 1, 2004; 24(11): 4824 - 4834. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q.-l. Wang, S. Chen, N. Esumi, P. K. Swain, H. S. Haines, G. Peng, B. M. Melia, I. McIntosh, J. R. Heckenlively, S. G. Jacobson, et al. QRX, a novel homeobox gene, modulates photoreceptor gene expression Hum. Mol. Genet., May 15, 2004; 13(10): 1025 - 1040. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Wang, S. Xu, C. Rivolta, L. Y. Li, G.-H. Peng, P. K. Swain, C.-H. Sung, A. Swaroop, E. L. Berson, T. P. Dryja, et al. Barrier to Autointegration Factor Interacts with the Cone-Rod Homeobox and Represses Its Transactivation Function J. Biol. Chem., November 1, 2002; 277(45): 43288 - 43300. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. P. M. Cremers, J. A. J. M. van den Hurk, and A. I. den Hollander Molecular genetics of Leber congenital amaurosis Hum. Mol. Genet., May 15, 2002; 11(10): 1169 - 1176. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Chen, Q.-L. Wang, S. Xu, I. Liu, L. Y. Li, Y. Wang, and D. J. Zack Functional analysis of cone-rod homeobox (CRX) mutations associated with retinal dystrophy Hum. Mol. Genet., April 15, 2002; 11(8): 873 - 884. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
