The Human Poly(A)-binding Protein 1 Shuttles between the Nucleus and the Cytoplasm*
- From the Human Retrovirus Section, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201
Abstract
We have studied the intracellular localization of poly(A)-binding protein 1 (PABP1) by indirect immunofluorescence as well as by tagging with the green fluorescent protein (GFP) in living cells. We show that PABP1 is able to enter the nucleus. Accumulation of PABP1 in the nuclei was observed upon transcription inhibition, suggesting that active transcription is required for PABP1 export. The nuclear import of PABP1 is an energy-dependent process since PABP1 fails to enter the nucleus upon ATP depletion and at low temperature. Transfection of PABP1 or PABP1-GFP resulted in heterogeneity of intracellular distribution of the protein. In the low expressing cells, PABP1 was localized in the cytoplasm, whereas in the high expressors, we observed accumulation of the protein in the nucleus. Nuclear PABP1 observed either after overexpression or after transcription inhibition was found in speckles and colocalized with splicing factor SC35. The ability of PABP1 to shuttle between nucleus and cytoplasm was also shown by heterokaryon formation upon cell fusion. Deletion mutagenesis showed that the minimal part of PABP1 retaining the ability to shuttle consists of the first two RNA-binding domains. This mutant interacted with poly(A) RNA with high affinity and accumulated in the nucleus. Deletion mutants exhibiting reduced RNA binding affinity did not accumulate in the nucleus. PABP1 has been proposed to participate at various steps of mRNA utilization. Our results suggest involvement of PABP1 in nuclear events associated with the formation and transport of mRNP to the cytoplasm and identify a new trafficking pattern for RNA-binding proteins.
Footnotes
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↵* This work was sponsored by the NCI, DHHS, National Institutes of Health, under contract with ABL.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ Both authors contributed equally to this work.
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↵§ Present address: Institute fur Klinische und Molekulare Virologie, Erlangen, Germany.
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↵¶ To whom correspondence should be addressed: ABL-Basic Research Program, P. O. Box B, Bldg. 535, Rm. 210, NCI-FCRDC, Frederick, MD 21702-1201. Tel.: 301-846-1474; Fax: 301-846-6368; E-mail:pavlakis{at}ncifcrf.gov.
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↵1 The abbreviations used are: PABP1, poly(A)-binding protein 1; GFP, green fluorescent protein; INS1, inhibitory/instability sequence 1; HIV-1, human immunodeficiency virus, type 1; LTR, long terminal repeat; nt, nucleotide(s); PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; RDR, rhodamine red; DRB, 5,6- dichlororibofuranosylbenzimidazole.
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↵2 R. Stauber, E. Afonina, and G. N. Pavlakis, unpublished data.
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↵3 S. Lee, M. Neumann, R. Stauber, A. Pause, S. Zhou, G. N. Pavlakis, and R. D. Klausner, submitted for publication.
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- Received October 3, 1997.
- Revision received January 15, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
