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Vol. 19, Issue 12, 5296-5308, December 2008

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eEF1A Is a Novel Component of the Mammalian Nuclear Protein Export Machinery

Mireille Khacho^*, Karim Mekhail^*,, Karine Pilon-Larose^*, Arnim Pause, Jocelyn Côté^*, and Stephen Lee^*

*Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, K1H 8M5, Canada; and McGill Cancer Center and Department of Biochemistry, McGill University, Montreal, Quebec, H3G 1Y6, Canada

Submitted June 4, 2008; Revised August 26, 2008; Accepted September 10, 2008
Monitoring Editor: A. Gregory Matera

The cytoplasmic translation factor eEF1A has been implicated in the nuclear export of tRNA species in lower eukaryotes. Here we demonstrate that eEF1A plays a central role in nuclear export of proteins in mammalian cells. TD-NEM (transcription-dependent nuclear export motif), a newly characterized nuclear export signal, mediates efficient nuclear export of several proteins including the von Hippel-Lindau (VHL) tumor suppressor and the poly(A)-binding protein (PABP1) in a manner that is dependent on ongoing RNA polymerase II (RNA PolII)-dependent transcription. eEF1A interacts specifically with TD-NEM of VHL and PABP1 and disrupting this interaction, by point mutations of key TD-NEM residues or treatment with actinomycin D, an inhibitor of RNA PolII-dependent transcription, prevents assembly and nuclear export. siRNA-induced knockdown or antibody-mediated depletion of eEF1A prevents in vivo and in vitro nuclear export of TD-NEM–containing proteins. Nuclear retention experiments and inhibition of the Exportin-5 pathway suggest that eEF1A stimulates nuclear export of proteins from the cytoplasmic side of the nuclear envelope, without entering the nucleus. Together, these data identify a role for eEF1A, a cytoplasmic mediator of tRNA export in yeast, in the nuclear export of proteins in mammalian cells. These results also provide a link between the translational apparatus and subcellular trafficking machinery demonstrating that these two central pathways in basic metabolism can act cooperatively.

Present address: Department of Cell Biology, Harvard Medical School, Harvard University, 240 Longwood Avenue, Boston, MA 02115.

Address correspondence to: Stephen Lee (slee{at}uottawa.ca)

Abbreviations used: ActD, actinomycin D; CRM1, Chromosome Region Maintenance (exportin 1); Dbp5, the DEAD box RNA helicase Rat8p/Dbp5p; eEF1A, eukaryotic translation elongation factor 1 alpha; FLIP, fluorescence loss in photobleaching; LMB, leptomycin B; NES, nuclear export sequence; NLS, nuclear localization signal; NoDS^H+, nucleolar detention signal regulated by [H⁺]; PABP1, poly(A)-binding protein 1; PK, pyruvate kinase; TD-NEM, transcription-dependent nuclear export motif; VHL, von Hippel-Lindau.