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Mol. Cell. Biol. 22 (15): 5319-5336

Copyright © 2002 by the American Society for Microbiology. All rights reserved.

Oxygen-Dependent Ubiquitination and Degradation of Hypoxia-Inducible Factor Requires Nuclear-Cytoplasmic Trafficking of the von Hippel-Lindau Tumor Suppressor Protein

Isabelle Groulx and Stephen Lee*

Department of Cellular and Molecular Medicine and Kidney Research Center, Faculty of Medicine, University of Ottawa, Ottawa, K1H 8M5 Ontario, Canada

Received for publication 8 February 2002. Revision received 8 March 2002. Accepted for publication 30 April 2002.

Abstract: It is becoming increasingly evident that the degradation of nuclear proteins requires nuclear-cytoplasmic trafficking of both the substrate proteins, as well as the E3 ubiquitin-ligases. Here, we show that nuclear-cytoplasmic trafficking of the von Hippel-Lindau tumor suppressor protein (VHL) is required for oxygen-dependent ubiquitination and degradation of the alpha subunits of hypoxia-inducible factor (HIF-{alpha}). VHL engages in a constitutive transcription-sensitive nuclear-cytoplasmic shuttle unaffected by oxygen tension or levels of nuclear substrate HIF-{alpha}. Ubiquitinated forms of HIF-{alpha}, as well as VHL/ubiquitinated HIF-{alpha} complexes, are found solely in the nuclear compartment of normoxic or reoxygenated VHL-competent cells. HIF-{alpha} localizes exclusively in the nucleus of hypoxic cells but is exported to the cytoplasm upon reoxygenation. Oxygen-dependent nuclear ubiquitination and nuclear export of HIF-{alpha} can be prevented by treatment with an HIF-specific prolyl hydroxylase inhibitor. Treatment with inhibitors of RNA polymerase II activity, which interfere with the ability of VHL to engage in nuclear export, also prevents cytoplasmic accumulation of HIF-{alpha} in reoxygenated cells. This caused a marked increase in the HIF-{alpha} half-life without affecting its nuclear ubiquitination. We present a model by which VHL-mediated ubiquitination of HIF-{alpha} and its subsequent degradation are dependent upon dynamic nuclear-cytoplasmic trafficking of both the E3 ubiquitin-ligase and the nuclear substrate protein.

* Corresponding author. Mailing address: Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, K1H 8M5 Ontario, Canada. Phone: (613) 562-5800, x8385. Fax: (613) 562-5636. E-mail: slee{at}

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