The purified recombinant VHL tumor suppressor complex activates HIF1α ubiquitination in vitro. (A) An aliquot of recombinant His-HPC4-HIF1α used as substrate in the ubiquitination reactions of C and D was subjected to 8% SDS/PAGE, and proteins were visualized by Coomassie staining. His-HPC4-HIF1α was expressed in Sf21 cells and purified by Ni²⁺-agarose chromatography. (B) The recombinant VHL complex was purified as described in Materials and Methods from lysates of insect cells infected with baculoviruses encoding His-VHL, Cul2, Elongin B, Elongin C, and myc-Rbx1. Aliquots of TSK DEAE-NPR column fractions were subjected to 13% SDS/PAGE, and proteins were visualized by Coomassie staining (Top). Aliquots of TSK DEAE-NPR column fractions were subjected to 8% or 13% SDS/PAGE, and proteins were transferred to Hybond P membranes and visualized by Western blotting as described in Materials and Methods, using the antibodies indicated in the figure (Bottom). EloB, Elongin B; EloC, Elongin C. (C) Aliquots of TSK-DEAE-NPR column fractions indicated in the figure were assayed as described in Materials and Methods for the ability to activate HIF1α ubiquitination. Reactions contained ≈50 ng of the His-HPC4-HIF1α shown in A. Reaction products were subjected to 8% SDS/PAGE, and GST-ubiquitin-HIF1α conjugates (GST-Ub-HIF1α) were transferred to Hybond P membranes and visualized by Western blotting as described in Materials and Methods, using anti-HPC4 antibodies. (D) Aliquots of TSK-DEAE-NPR column fraction 25 were assayed as described in Materials and Methods for the ability to activate HIF1α or Cln2 ubiquitination activity in the presence and absence of Uba1, hUbc5a, GST-ubiquitin (GST-Ub), and ATP. Reactions contained ≈50 ng of either the His-HPC4-HIF1α shown in A or about 50 ng of the phosphorylated Cln2 complex. Reaction products were subjected to 8% SDS/PAGE and transferred to Hybond P membranes and visualized by Western blotting as described in Materials and Methods, using anti-HPC4 or anti-HA antibodies. (E) Aliquots of TSK-DEAE-NPR column fraction 25 were assayed as described in Materials and Methods for the ability to activate HIF1α ubiquitination activity in the presence of Uba1, GST-ubiquitin (GST-Ub), ATP, and approximately equimolar amounts of the E2 ubiquitin-conjugating enzymes shown in the figure (≈100 ng hUbc5a, ≈200 ng hUbc3, ≈100 ng mE2-21K, ≈200 ng mE2-35K, ≈150 ng mE2-24K). Reactions contained ≈50 ng of the His-HPC4-HIF1α shown in A. Reaction products were subjected to 8% SDS/PAGE, and GST-ubiquitin-HIF1α conjugates (GST-Ub-HIF1α) were transferred to Hybond P membranes and visualized by Western blotting as described in Materials and Methods, using anti-HPC4 antibodies.

VHL mutants that do not bind stably to Elongins B and C do not assemble into the VHL complex and do not detectably activate HIF1α ubiquitination. (A) Total cell lysates from Sf21 cells infected with the baculoviruses indicated in the figure were subjected to 8% or 13% SDS/PAGE, and proteins were transferred to Hybond P membranes and visualized by Western blotting as described in Materials and Methods, using the antibodies indicated in the figure. (B) The cell lysates of A were subjected to immunoprecipitations with anti-VHL antibodies as described in Materials and Methods. The immunoprecipitates were subjected to 8% or 13% SDS/PAGE, and proteins were transferred to Hybond P membranes and visualized by Western blotting as described in Materials and Methods, using the antibodies indicated in the figure. (C) The cell lysates of A were subjected to immunoprecipitations with anti-VHL antibodies as described in Materials and Methods. The immunoprecipitates were assayed for the ability to activate HIF1α ubiquitination as described in Materials and Methods. Reaction products were subjected to 8% SDS/PAGE, and GST-ubiquitin-HIF1α conjugates (GST-Ub-HIF1α) were transferred to Hybond P membranes and visualized by Western blotting as described in Materials and Methods, using anti-HIF1α antibodies. EloB, Elongin C; EloC, Elongin C.

A VHL mutant lacking a portion of the β domain is defective in HIF1α binding and ubiquitination. (A) Total cell lysates or anti-HIF1α immunoprecipitates from the indicated baculovirus-infected Sf21 cells were immunoblotted with anti-Flag antibody. (B) Sf21 cells were coinfected with baculoviruses encoding Cul2, Elongin B, Elongin C, His-myc-Rbx1, and either His-VHL-Flag or His-VHL-Flag[117–213]. Recombinant VHL complexes were purified as described in Materials and Methods. After TSK DEAE-NPR HPLC, aliquots of peak fractions from were subjected to 13% SDS/PAGE, and proteins were visualized by Coomassie staining. (C) Aliquots of the TSK DEAE-NPR fractions shown in B were assayed as described in Materials and Methods for the ability to activate HIF1a ubiquitination. Reactions contained ≈50 ng of the His-HPC4-HIF1a shown in Fig. 1A. The reaction products were subjected to 8% SDS/PAGE, transferred to Hybond P membranes, and visualized by Western blotting using anti-HPC4 antibody. H, His; Fl, Flag.

All subunits of the VHL complex are required for maximal activation of HIF1α ubiquitination. (A) Total cell lysates from Sf21 cells infected with the baculoviruses indicated in the figure were subjected to 8% or 13% SDS/PAGE, and proteins were transferred to Hybond P membranes and visualized by Western blotting as described in Materials and Methods, using the antibodies indicated in the figure. (B) The cell lysates of A were subjected to immunoprecipitations with anti-VHL antibodies as described in Materials and Methods. The immunoprecipitates were subjected to 8% or 13% SDS/PAGE, and proteins were transferred to Hybond P membranes and visualized by Western blotting as described in Materials and Methods, using the antibodies indicated in the figure. (C) The cell lysates of A were subjected to immunoprecipitations with anti-VHL antibodies as described in Materials and Methods. The immunoprecipitates were assayed for the ability to activate HIF1α ubiquitination as described in Materials and Methods. Reaction products were subjected to 8% SDS/PAGE, and GST-ubiquitin-HIF1α conjugates (GST-Ub-HIF1α) were transferred to Hybond P membranes and visualized by Western blotting as described in Materials and Methods, using anti-HIF1α antibodies. EloB, Elongin B; EloC, Elongin C.

Interaction of HIF1α with VHL mutants VHL[1–155] and VHL[L158P]. (Upper) Total cell lysates from Sf21 cells infected with the baculoviruses indicated in the figure were subjected to 8% or 13% SDS/PAGE, and proteins were transferred to Hybond P membranes and visualized by Western blotting as described in Materials and Methods, using the antibodies indicated in the figure. (Middle and Lower) Cell lysates were subjected to immunoprecipitations with anti-VHL or anti-Flag antibodies as described in Materials and Methods. The immunoprecipitates were subjected to 8% or 13% SDS/PAGE, and proteins were transferred to Hybond P membranes and visualized by Western blotting as described in Materials and Methods, using the antibodies indicated in the figure.