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Molecular and Cellular Biology, August 2003, p. 5308-5319, Vol. 23, No. 15
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.15.5308-5319.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Overlapping Signals for Protein Degradation and Nuclear Localization Define a Role for Intrinsic RAG-2 Nuclear Uptake in Dividing Cells

Ashley E. Ross, Milena Vuica, and Stephen Desiderio^*

Department of Molecular Biology and Genetics and Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Received 3 March 2003/ Returned for modification 10 April 2003/ Accepted 16 May 2003

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 G₁-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.

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* Corresponding author. Mailing address: Johns Hopkins University School of Medicine, 725 N. Wolfe St., PCTB 701, Baltimore, MD 21205. Phone: (410) 955-4735. Fax: (410) 955-9124. E-mail: sdesider{at}jhmi.edu.

Present address: Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205.

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Molecular and Cellular Biology, August 2003, p. 5308-5319, Vol. 23, No. 15
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.15.5308-5319.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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