Volume 4, Issue 6, Pages 1051-1061 (December 1999) Formation of the VHL–Elongin BC Tumor Suppressor Complex Is Mediated by the Chaperonin TRiC  Douglas E Feldman, Vanitha Thulasiraman, Raul G Ferreyra, Judith Frydman  Molecular Cell  Volume 4, Issue 6, Pages 1051-1061 (December 1999) DOI: 10.1016/S1097-2765(00)80233-6

Figure 1 A Large Chaperone Complex Containing TRiC/CCT Associates with Unassembled VHL in Mammalian Cells (A) VHL associates with the cytosolic chaperonin TRiC/CCT and Hsp/Hsc70 in vivo. Human embryonic kidney (HEK) 293 cells expressing Flag-tagged VHL (lanes 2 and 3) or containing the backbone vector plasmid (lane 1) were labeled with [35S]methionine, lysed, and analyzed by immunoprecipitation with anti-TRiC (lanes 1 and 2) or anti-Flag (lane 3) antibodies. Bound proteins were resolved by 12% SDS-PAGE. TRiC, Hsp/Hsc70, and VHL are indicated; the 12% gel does not resolve elongin BC from the front. (B) TRiC–VHL levels are reduced by coexpression of VHL with elongin BC. VHL was expressed in HEK293 in the absence (lane 2) or presence (lane 3) of additional elongin B and elongin C. Cells were labeled as in (A) and subjected to immunoprecipitation with anti-TRiC (lane 1) or anti-Flag (lanes 2–4) antibodies. Samples were analyzed by 12% and 15% SDS-PAGE (upper and lower panels, respectively) to visualize elongin B and C. Endogenous human elongin B had a slightly slower mobility than the transfected rat elongin B (see double arrow). (C and D) VHL participates in two mutually exclusive complexes. Lysates prepared as in (B), from VHL or VHL plus elongin BC–expressing cells, were analyzed on a Superose 6 gel filtration column. The presence of VHL in individual column fractions (1 ml) was examined by immunoprecipitation and 18% SDS-PAGE analysis (shown in [C] for VHL plus elongin BC). VHL was quantitated using a Bio-Rad Phosphorimager (D). Molecular Cell 1999 4, 1051-1061DOI: (10.1016/S1097-2765(00)80233-6)

Figure 6 Deletion of VHL Exon 2 Abolishes TRiC Binding and Formation of Correctly Folded VBC In Vivo (A) VHL exon 2 is essential for interaction with TRiC. Wild-type VHL (lane 2), VHL–Δex2 (lane 3), or control vector (lanes 1 and 4) were expressed in 786-O VHL−/− cells, and the interaction of cellular proteins with VHL (lanes 2–4) was analyzed by immunoprecipitation with anti-Flag antibodies, followed by 12% SDS-PAGE. An immunoprecipitation with TRiC (lane 1) is included for comparison. The inability of VHL–Δex2 to interact with TRiC was confirmed by immunoblot analysis of the VHL immunoprecipitates. (B) VHL exon 2 is essential for proper assembly with elongin BC. Wild-type VHL (lanes 1 and 2) or VHL–Δex2 (lanes 3 and 4) were transfected in HEK293 cells with or without additional elongin BC and analyzed by immunoprecipitation followed by SDS-PAGE. The migrations of human (hB) and rat (rB) elongin B are indicated. (C) VHL–Δex2 is in a protease-sensitive conformation in vivo. Lysates prepared from [35S]methionine HEK-293 cells expressing VBC (lanes 1–3) or VHL–Δex2–BC (lanes 4–6) were subjected to treatment with thermolysin (30 μg/ml, Th) or chymotrypsin (10 μg/ml, Ch) as described in the Experimental Procedures. Molecular Cell 1999 4, 1051-1061DOI: (10.1016/S1097-2765(00)80233-6)

Figure 2 The High Molecular Weight VHL–Chaperone Complex Mediates the ATP-Dependent Assembly of VBC The experimental design is shown in the scheme. The high molecular weight VHL–chaperone complex was purified from 35S-labeled HEK293 cells by gel filtration, as in Figure 1D. Fractions eluting at 11.5–13 ml, containing VHL bound to TRiC and Hsp/Hsc70 (lane 1) were pooled and incubated for 10 min at 30°C as indicated, in the presence or absence of 1 mM ATP/2 mM MgCl2 and 3 μM BC-His6. To assess whether these treatments dissociated the chaperone–substrate complex, the VHL-bound proteins were analyzed by anti-Flag immunoprecipitation (lanes 1–4). Assembly of VHL into the VBC–His6 complex was determined by binding of the 35S-labeled VHL to Co2+-agarose beads (lanes 5–8). Molecular Cell 1999 4, 1051-1061DOI: (10.1016/S1097-2765(00)80233-6)

Figure 4 Tumor-Derived VHL Mutation L158P Remains Bound to TRiC (A and B) Effect of VHL point mutation L158P on TRiC binding and VBC assembly in vivo and in vitro. (A) In vitro analysis. Flag-tagged wild-type VHL (WT) or the tagged point mutant L158P-VHL were translated in rabbit reticulocyte lysate (lanes 2 and 3). Binding to elongin BC was examined after incubation with in vitro–translated elongin BC (lane 1) for 30 min at 30°C followed by immunoprecipitation with anti-Flag antibody (lanes 4–6). The interaction with TRiC was assessed by immunoprecipitation (lanes 7–9). The totals shown (lanes 1–3) are equal to 10% of the input to the immunoprecipitations. (B) In vivo analysis. Flag-tagged forms of wild-type VHL (lanes 3 and 4) and L158P-VHL (lanes 1 and 2) were expressed in HEK-293 cells with or without additional elongin BC. Cells were labeled with [35S]methionine, and VHL-bound proteins were analyzed by immunoprecipitation. (C) L158P-VHL is not released from the complex with TRiC in vivo. The association of L158P-VHL with cellular proteins was analyzed by gel filtration chromatography as described for Figure 1C and Figure 1D. The trace of wild-type VHL (from Figure 1C and Figure 1D) is included for comparison. Both VHL variants were coexpressed with additional elongin BC. (D) L158P-VHL remains in a protease-sensitive conformation. The protease susceptibility of VBC (lanes 1–3) and L158P-VHL BC (lanes 4–6) was examined as described in the Experimental Procedures. Th, thermolysin; Ch, chymotrypsin. Molecular Cell 1999 4, 1051-1061DOI: (10.1016/S1097-2765(00)80233-6)

Figure 3 TRiC Is Required for Assembly of VHL into the VBC Complex (A) Immunoblot analysis of TRiC-depleted translation lysates. TRiC was immunodepleted from reticulocyte lysate by incubation with anti-TRiC antibodies (ΔTRiC, lane 3). Nonimmune serum was used as a control (NI, lane 2). Immunoblot analysis using a monoclonal anti-TCP1 antibody indicated that the efficiency of depletion was greater than 90%. RL, untreated lysate. (B) TRiC is required for assembly of the VBC complex. VHL was translated for 1 hr at 30°C in mock-depleted (lanes 1 and 2) or TRiC-depleted (lanes 3 and 4) lysates containing BC-His6 (5 μM). Where indicated, purified bovine TRiC was added to 0.35 μM. The amounts of total VHL translated (lanes 1–4) as well as VHL incorporated into VBC–His6 (lanes 5–8) were quantified by SDS-PAGE and Phosphorimager analysis. Immunodepletion of TRiC decreased the incorporation of newly translated VHL into VBC by 70% ± 15% relative to mock-depleted lysates (average of four experiments). Molecular Cell 1999 4, 1051-1061DOI: (10.1016/S1097-2765(00)80233-6)

Figure 5 A 55–Amino Acid Region of VHL Is Necessary and Sufficient for Binding to TRiC (A) The TRiC-binding site of VHL contains exon 2. N- and C-terminal deletions of VHL (scheme, “a”–“f”) and an internal deletion of exon 2 (Δex2, “g”) were translated in rabbit reticulocyte lysate in the presence of [35S]methionine for 30 min at 30°C (“a”–“g”, left panel). Binding to TRiC was examined by immunoprecipitation using anti-TRiC antibodies (“a”–“g”, right panel). The high amount of globin distorts the migration of “d” in the left panel. (B) The TRiC-binding site of VHL is transferable to a heterologous protein. Mouse DHFR (“a”) was fused in-frame to a VHL control region (“b”) or to the TRiC-binding site of VHL (“c”). Following translation in reticulocyte lysate (lanes 1–3), the samples were immunoprecipitated with either nonimmune (NI, lanes 4–6) or anti-TRiC (lanes 7–9) antibodies, as above. Molecular Cell 1999 4, 1051-1061DOI: (10.1016/S1097-2765(00)80233-6)