Endoplasmic reticulum/cytosolic localization of von hippel-lindau gene products is mediated by a 64-amino acid region

Alan R. Schoenfeld, Eliot J. Davidowitz, Robert D. Burk

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

Inactivation of the von Hippel-Lindau (VHL) tumor-suppressor gene causes both the familial cancer syndrome VHL disease and corresponding sporadic tumor types, including renal-cell carcinoma. Subcellular localization of VHL gene products was determined by indirect immunofluorescence. Both native and exogenously expressed VHL proteins displayed a cytoplasmic peri-nuclear immunostaining pattern, which co-localized with markers for the endoplasmic reticulum (ER). In addition, subcellular fractionation indicated that both native and exogenously expressed VHL products are found predominantly in the cytosolic compartment. Deletion analyses demonstrated that a 64-amino acid region of VHL (residues 114-177) is responsible for cytosolic as well as ER subcellular localization. Taken together, the immunostaining and biochemical fractionation studies suggest that VHL localizes to the cytosolic face of the ER. The relationship between VHL subcellular localization and VHL-associated ubiquitination was examined. Chimeric VHL-green fluorescent protein (GFP) products, which localized to the peri-nuclear region, were shown to undergo ubiquitination. VHL amino acids 114-177 were necessary and sufficient for this modification. Consistent with a role of VHL in ubiquitination, expression of VHL led to enhanced ubiquitination of cellular proteins, and amino acids 114-177 were also critical for this effect. Therefore, amino acids 114-177 were required for accurate VHL subcellular localization, ubiquitination of VHL-GFP products and VHL-dependent increases in cellular ubiquitination. Since mutations in this region of VHL are frequently detected in renal-cell carcinomas, these results suggest that proper VHL subcellular localization and associated ubiquitination functions may be necessary for VHL-mediated tumor suppression.

Original languageEnglish (US)
Pages (from-to)457-467
Number of pages11
JournalInternational Journal of Cancer
Volume91
Issue number4
DOIs
StatePublished - Feb 15 2001

Fingerprint

Ubiquitination
Endoplasmic Reticulum
Amino Acids
Genes
Green Fluorescent Proteins
Renal Cell Carcinoma
von Hippel-Lindau Disease
Neoplasms
Indirect Fluorescent Antibody Technique
Tumor Suppressor Genes
Proteins
Mutation

Keywords

  • Endoplasmic reticulum
  • Renal-cell carcinoma
  • Subcellular localization
  • Ubiquitination
  • Von Hippel-Lindau (VHL)

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Endoplasmic reticulum/cytosolic localization of von hippel-lindau gene products is mediated by a 64-amino acid region. / Schoenfeld, Alan R.; Davidowitz, Eliot J.; Burk, Robert D.

In: International Journal of Cancer, Vol. 91, No. 4, 15.02.2001, p. 457-467.

Research output: Contribution to journalArticle

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abstract = "Inactivation of the von Hippel-Lindau (VHL) tumor-suppressor gene causes both the familial cancer syndrome VHL disease and corresponding sporadic tumor types, including renal-cell carcinoma. Subcellular localization of VHL gene products was determined by indirect immunofluorescence. Both native and exogenously expressed VHL proteins displayed a cytoplasmic peri-nuclear immunostaining pattern, which co-localized with markers for the endoplasmic reticulum (ER). In addition, subcellular fractionation indicated that both native and exogenously expressed VHL products are found predominantly in the cytosolic compartment. Deletion analyses demonstrated that a 64-amino acid region of VHL (residues 114-177) is responsible for cytosolic as well as ER subcellular localization. Taken together, the immunostaining and biochemical fractionation studies suggest that VHL localizes to the cytosolic face of the ER. The relationship between VHL subcellular localization and VHL-associated ubiquitination was examined. Chimeric VHL-green fluorescent protein (GFP) products, which localized to the peri-nuclear region, were shown to undergo ubiquitination. VHL amino acids 114-177 were necessary and sufficient for this modification. Consistent with a role of VHL in ubiquitination, expression of VHL led to enhanced ubiquitination of cellular proteins, and amino acids 114-177 were also critical for this effect. Therefore, amino acids 114-177 were required for accurate VHL subcellular localization, ubiquitination of VHL-GFP products and VHL-dependent increases in cellular ubiquitination. Since mutations in this region of VHL are frequently detected in renal-cell carcinomas, these results suggest that proper VHL subcellular localization and associated ubiquitination functions may be necessary for VHL-mediated tumor suppression.",
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