Abstract
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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 8991-9000 |
| Number of pages | 10 |
| Journal | Journal of Biological Chemistry |
| Volume | 275 |
| Issue number | 12 |
| DOIs | |
| State | Published - Mar 24 2000 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Ran-mediated nuclear export of the von Hippel-Lindau tumor suppressor protein occurs independently of its assembly with cullin-2. / Groulx, Isabelle; Bonicalzi, Marie Eve; Lee, Stephen.
In: Journal of Biological Chemistry, Vol. 275, No. 12, 24.03.2000, p. 8991-9000.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Ran-mediated nuclear export of the von Hippel-Lindau tumor suppressor protein occurs independently of its assembly with cullin-2
AU - Groulx, Isabelle
AU - Bonicalzi, Marie Eve
AU - Lee, Stephen
PY - 2000/3/24
Y1 - 2000/3/24
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.275.12.8991
DO - 10.1074/jbc.275.12.8991
M3 - Article
VL - 275
SP - 8991
EP - 9000
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 12
ER -