Ran-mediated nuclear export of the von Hippel-Lindau tumor suppressor protein occurs independently of its assembly with cullin-2.
Inactivating mutations of the von Hippel-Lindau (VHL) tumor suppressor gene cause the VHL cancer syndrome and sporadic renal clear cell carcinoma. VHL engages in a nucleocytoplasmic shuttle, which is required for its function. Here, we pursue our investigation to identify mechanisms by which VHL-green fluorescent protein (VHL-GFP) is exported from the nucleus. We show that nuclear export of VHL-GFP in living cells requires ongoing RNA polymerase II activity, and is mediated by mechanisms that are temperature-sensitive and energy-dependent. In vitro nuclear export of VHL-GFP is inhibited by nuclear pore-specific lectins, requires ATP hydrolysis and polyadenylated mRNAs, and occurs with kinetics that are similar to those of proteins containing a nuclear export signal. Biochemical fractionation has revealed that nuclear export of VHL-GFP occurs by way of a Ran-dependent pathway. Size exclusion column chromatography and deletion mutant analysis suggest that VHL-GFP does not require assembly with one of its associated proteins, cullin-2, to engage in nuclear export. These results demonstrate that nuclear export of VHL-GFP is Ran-mediated and ATP hydrolysis-dependent. They also suggest that sequences outside the elongin C binding box may function as a nuclear export domain, potentially providing a novel role for this region of VHL frequently mutated in renal cell carcinoma.
Groulx
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Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada.
Bonicalzi
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Lee
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D000255:Adenosine Triphosphate / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D002451:Cell Compartmentation; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D002474:Cell-Free System; D044842:Cullin Proteins; D016147:Genes, Tumor Suppressor; D049452:Green Fluorescent Proteins; D006153:Guanosine Diphosphate / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D037102:Lectins / Q000494:pharmacology; D008025:Ligases; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009685:Nuclear Envelope / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012319:RNA Polymerase II / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D020931:ran GTP-Binding Protein / Q000378:metabolism*
C105153:CUL2 protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D044842:Cullin Proteins; D037102:Lectins; D008164:Luminescent Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; D006153:Guanosine Diphosphate; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D012319:RNA Polymerase II; D020931:ran GTP-Binding Protein; D008025:Ligases; C497446:VHL protein, human