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

Inactivation of the von Hippel-Lindau (VHL) tumor-suppressor gene causes bo th the familial cancer syndrome VHL disease and corresponding sporadic tumo r types, including renal-cell carcinoma. Subcellular localization of VHL ge ne products was determined by indirect immunofluorescence. Both native and exogenously expressed VHL proteins displayed a cytoplasmic peri-nuclear imm unostaining pattern, which co-localized with markers for the endoplasmic re ticulum (ER). In addition, subcellular fractionation indicated that both na tive 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 biochemic al fractionation studies suggest that VHL localizes to the cytosolic face o f the ER, The relationship between VHL subcellular localization and VHL-ass ociated 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 sufficie nt 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 ac ids 114-177 were required for accurate VHL subcellular localisation, ubiqui tination of VHL-GFP products and VHL-dependent increases in cellular ubiqui tination. Since mutations in this region of VHL are frequently detected in renal-cell carcinomas, these results suggest that proper VHL subcellular lo calization and associated ubiquitination functions may be necessary for VHL -mediated tumor suppression. (C) 2001 Wiley-Liss, Inc.