Overlapping signals for protein degradation and nuclear localization define a role for intrinsic RAG-2 nuclear uptake in dividing cells.
Expression of the recombinase proteins RAG-1 and RAG-2 is discordant: while RAG-1 is relatively long lived, RAG-2 is degraded periodically at the G(1)-S transition. Destruction of RAG-2 is mediated by a conserved interval in the recombination-dispensable region. The need for RAG-2 to reaccumulate in the nucleus at each cell division suggested the existence of an intrinsic RAG-2 nuclear localization signal (NLS). RAG-1 or RAG-2, expressed individually, is a nuclear protein. A screen for proteins that bind the recombination-dispensable region of RAG-2 identified the nuclear transport protein Importin 5. Mutation of residues 499 to 508 in RAG-2 abolished Importin 5 binding, nuclear accumulation, and periodic degradation of RAG-2. The Importin 5 binding site overlaps an NLS, defined by mutagenesis. RAG-1 rescued the localization of degradation-defective, RAG-2 NLS mutants; this required an intact RAG-1 NLS. Mutations in RAG-2 that abolish intrinsic nuclear accumulation but spare periodic degradation impaired recombination in cycling cells; induction of quiescence restored recombination to wild-type levels. Recombination defects were correlated with a cell cycle-dependent defect in the ability of RAG-1 to rescue localization of the RAG-2 mutants. These results suggest that the intrinsic RAG-2 NLS functions in the nuclear uptake of RAG-2 following its reexpression in cycling cells.
Ross
Ashley E
AE
Department of Molecular Biology and Genetics and Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA.
Vuica
Milena
M
Desiderio
Stephen
S
D016475:3T3 Cells; D021581:Active Transport, Cell Nucleus; D000818:Animals; D001665:Binding Sites; D002453:Cell Cycle; D002455:Cell Division; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D016193:G1 Phase; D016654:Genes, RAG-1 / Q000235:genetics*; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic; D016196:S Phase; D014162:Transfection; D045567:VDJ Recombinases; D028901:alpha Karyopherins / Q000378:metabolism
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C423142:KPNA1 protein, human; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; C499311:RAG2 protein, human; C499308:Rag2 protein, mouse; C064655:V(D)J recombination activating protein 2; D028901:alpha Karyopherins; C064658:RAG-1 protein; D004247:DNA; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases