The study investigates the mechanism by which the von Hippel-Lindau (VHL) tumor suppressor protein regulates the hypoxia-inducible factor-1α (HIF-1α) under normoxic conditions. VHL is shown to mediate the ubiquitylation and proteasomal degradation of HIF-1α through interaction with the core of the oxygen-dependent degradation domain of HIF-1α. The region of VHL mediating this interaction overlaps with a putative macromolecular binding site observed in the crystal structure of VHL, which also represents a mutational hotspot in tumors. The VHL binding site within HIF-1α overlaps with one of the minimal transactivation domains. Protection of HIF-1α from VHL-mediated degradation requires both nuclear translocation of HIF-1α and an intranuclear hypoxia-dependent signal. Stabilization of HIF-1α protein levels alone does not bypass the need for the hypoxic signal to generate the transactivation response. The findings suggest that VHL regulates HIF-1α function by targeting it for ubiquitylation and proteasomal degradation, with the hypoxic signal playing a crucial role in protecting HIF-1α from this degradation.The study investigates the mechanism by which the von Hippel-Lindau (VHL) tumor suppressor protein regulates the hypoxia-inducible factor-1α (HIF-1α) under normoxic conditions. VHL is shown to mediate the ubiquitylation and proteasomal degradation of HIF-1α through interaction with the core of the oxygen-dependent degradation domain of HIF-1α. The region of VHL mediating this interaction overlaps with a putative macromolecular binding site observed in the crystal structure of VHL, which also represents a mutational hotspot in tumors. The VHL binding site within HIF-1α overlaps with one of the minimal transactivation domains. Protection of HIF-1α from VHL-mediated degradation requires both nuclear translocation of HIF-1α and an intranuclear hypoxia-dependent signal. Stabilization of HIF-1α protein levels alone does not bypass the need for the hypoxic signal to generate the transactivation response. The findings suggest that VHL regulates HIF-1α function by targeting it for ubiquitylation and proteasomal degradation, with the hypoxic signal playing a crucial role in protecting HIF-1α from this degradation.