The Hsp70 and TRiC/CCT chaperone systems cooperate in vivo to assemble the von Hippel-Lindau tumor suppressor complex

Abstract

The degree of cooperation and redundancy between different chaperones is an important problem in understanding how proteins fold in the cell. Here we use the yeast Saccharomyces cerevisiae as a model system to examine in vivo the chaperone requirements for assembly of the von Hippel-Lindau protein (VHL)-elongin BC (VBC) tumor suppressor complex. VHL and elongin BC expressed in yeast assembled into a correctly folded VBC complex that resembles the complex from mammalian cells. Unassembled VHL did not fold and remained associated with the cytosolic chaperones Hsp70 and TRiC/CCT, in agreement with results from mammalian cells. Analysis of the folding reaction in yeast strains carrying conditional chaperone mutants indicates that incorporation of VHL into VBC requires both functional TRiC and Hsp70. VBC assembly was defective in cells carrying either a temperature-sensitive ssa1 gene as their sole source of cytosolic Hsp70/SSA function or a temperature-sensitive mutation in CCT4, a subunit of the TRiC/CCT complex. Analysis of the VHL-chaperone interactions in these strains revealed that the cct4ts mutation decreased binding to TRiC but did not affect the interaction with Hsp70. In contrast, loss of Hsp70 function disrupted the interaction of VHL with both Hsp70 and TRiC. We conclude that, in vivo, folding of some polypeptides requires the cooperation of Hsp70 and TRiC and that Hsp70 acts to promote substrate binding to TRiC.

FIG. 1.

Expression and assembly of correctly folded VBC complex in S. cerevisiae. (A) The scheme shows the expression strategy for VHL and the elongin BC proteins. Polyhistidine-tagged VHL (V) was expressed under control of either galactose or copper-inducible promoters. The myc-tagged elongin BC proteins (in a separate plasmid) were under galactose-inducible control; one version of the plasmid contained myc-elongin B alone (designated B), and the other contained both myc-elongin B and myc-elongin C (designated C). The selection markers are also indicated. (B) VHL and the elongin BC proteins are expressed in yeast. Lysates (50 μg) were prepared from yeast expressing His₆-VHL (V), His₆-VHL plus myc-elongin B (VB), His₆-VHL plus myc-elongins B and C (VBC), myc-elongins B and C (BC), myc-elongin B (B), or empty vectors (−); separated by SDS-PAGE; and immunoblotted with antibodies against VHL, elongin B, elongin C, or the myc epitope tag. (C) Formation of a stable VHL-elongin B-elongin C complex. Using antibodies against the myc epitope tag followed by immunoblot detection (Imm. Blot) of VHL, the association between different components of VBC in yeast was monitored by immunoprecipitation (Ipp.) of the elongin proteins. Lysates from yeast transformed with empty vectors (−), V, VBC, VB, or BC (as indicated) were used in this analysis. VHL was associated with elongins only in lysates containing all three components of VBC. The heavy and light chains from the anti-myc antibodies are indicated (HC and LC, respectively). (D) VHL in VBC is in a protease-resistant conformation. Lysates prepared from yeast transformed with VHL alone (V) or with VBC were treated with thermolysin and analyzed by SDS-PAGE and immunoblotting with VHL-specific antibodies. (E) Nondenaturing gel analysis of VHL-containing complexes. The complexes formed by VHL in yeast expressing either VHL with elongin BC (VBC; lane 1) or VHL alone (V; lane 2) were compared with the VBC complexes expressed in mammalian cells (lane 3). Lysates containing approximately equivalent levels of VHL were separated by nondenaturing PAGE, and VHL was detected by immunoblot analysis with specific antibodies. The slight differences in migration of the VBC complexes from different sources are caused by differences in the epitope tags of the components of the complexes (N-terminal polyhistidine-tagged VHL in yeast and FLAG-tagged VHL in mammalian cells). The origin of the gel is indicated (ori). The asterisk indicates slowly migrating putative complexes between VHL and endogenous yeast components.

FIG. 2.

Association of VHL with molecular chaperones in S. cerevisiae. (A) VHL-containing complexes immunoisolated from ³⁵S-labeled lysates. Yeast cells expressing VHL alone (V; lane 1), VHL and elongin BC (VBC; lane 2), or elongin BC (BC; lane 3) were metabolically labeled with [³⁵S]methionine. Following lysis, equal amounts of total counts (10⁶ cpm) were immunoprecipitated using VHL-specific antibodies and analyzed by SDS-PAGE and autoradiography. The positions of VHL and the elongin BC bands are indicated. The position of endogenous yeast proteins specifically associated with VHL are also indicated; the bracket indicates a set of bands with molecular masses similar to those of the components of the TRiC/CCT complex, whereas the asterisk indicates two bands migrating around 70 to 80 kDa. (B to D) Size exclusion chromatography analysis of VHL complexes in VBC lysates. (B) Elution profile of VHL, elongin BC, the Hsp70 Ssa1/2p, and TRiC/CCT following fractionation on a Superose 6 column. The graph shows the elution profile of VHL quantitated by densitometric analysis of immunoblots. (C) Association of VHL with the chaperonin TRiC/CCT. High-molecular-weight fractions (10 to 13 ml) of a Superose 6 fractionation of VBC lysates (as described for panel B) or BC lysates (as negative controls) were subjected to immunoprecipitation with VHL-specific antibodies followed by immunodetection (using antibodies directed against subunit CCT1) of TRiC. TRiC was only detected in the high-molecular-weight fractions of the VHL-containing lysates. The IgG heavy chain (HC) is indicated. (D) Association of VHL with the Hsp70 Ssa1/2p. Lysates and Superose 6 column fractions (pooled as indicated) were immunoprecipitated using a VHL-directed antibody followed by immunoblot detection of Ssa1/2p. A lysate from cells transformed with vector alone was used as a control.

FIG. 3.

The cytosolic Hsp70 Ssa1/2p is required for VHL folding and VBC assembly. (A) VHL is not incorporated into VBC in cells lacking Hsp70/Ssa1/2p function. VHL and elongin BC were expressed in SSA1wt and ssa1ts cells. Formation of VBC in SSA1wt (lanes 1 and 3) and ssa1ts (lanes 2 and 4) was monitored by immunoprecipitation (Ipp.) of myc-elongin BC-associated proteins (lanes 3 and 4). The top panel shows the results of immunoblot detection (Imm. Blot) of VHL; the bottom panel shows the results of immunoblot detection of elongin BC. Lanes 1 and 2 (Total) represent 10% of the amount of input lysate used in the immunoprecipitations. The heavy and light IgG chains are indicated (HC and LC, respectively). (B) VHL is in a protease-sensitive conformation in the ssa1ts cells. Lysates from VBC-expressing cells (SSA1wt, lanes 1 to 3; ssa1ts, lanes 4 to 6) were treated with thermolysin for the indicated times, and VHL was detected by immunoblot analysis. The graph shows the fraction of VHL remaining at each time point (expressed as percentage of initial amount) as quantified by densitometry. Results are representative of three similar experiments.

FIG. 4.

The cytosolic chaperonin TRiC/CCT is required for VHL folding and VBC assembly. (A and B) TRiC/CCT-mediated folding is impaired in the anc2-1/cct4ts cells. TRiC/CCT function in mediation of folding of endogenous actin was monitored according to binding to DNase I beads (A) and formation of protease-resistant actin (B). (A) Actin binding to DNase I-Sepharose beads examined in lysates of the cct4ts strain (lanes 1 and 3) and its isogenic wild-type strain (lanes 2 and 4). The binding assay was performed as described previously (15), and actin was detected by immunoblot analysis (Imm. Blot). Lanes 1 and 2 show the results for 10% input lysate; lanes 3 and 4 show the results for actin bound to DNase I-Sepharose beads. (B) Actin from cct4ts cells is in a protease-sensitive conformation. CCT4wt (lanes 1 to 3) and cct4ts (lanes 4 to 6) lysates were treated with proteinase K for the indicated times, and actin protein was detected by immunoblot analysis. The graph shows the fraction of actin remaining at each time point (expressed as percentage of initial amount) as quantified by densitometry. Results are representative of four similar experiments. (C and D) VHL folding and assembly are impaired in cct4ts cells. (C) VBC formation in the CCT4wt (lanes 1, 3, and 5) and cct4ts (lanes 2, 4, and 6) strains was examined by coimmunoprecipitation (Ipp.) of VHL with the elongin BC proteins (lanes 3 and 4). Lanes 1 and 2 (Total) represent 10% of the amount of input lysate used in the immunoprecipitations. Nonimmune mouse antibodies were used as negative controls (lanes 5 and 6). The top panel shows results for immunoblot detection of VHL; the bottom panel shows results for immunoblot detection of elongin BC. IgG heavy and light chains from the immunoprecipitations are indicated (HC and LC, respectively). (D) VHL is in a protease-sensitive conformation in the cct4ts cells. Lysates from VBC-expressing cells (CCT4wt, lanes 1 to 3; cct4ts, lanes 4 to 6) were treated with thermolysin for the indicated times, and VHL was detected by immunoblot analysis. The graph shows the fraction of VHL remaining at each time point (expressed as percentage of initial amount) as quantified by densitometry. Results are representative of six similar experiments.

FIG. 5.

Association of VHL with TRiC requires Hsp70 function. (A) Size exclusion chromatography analysis of VHL complexes in cells defective for TRiC and Hsp70 function. VBC lysates were prepared following expression in wild-type, cct4ts, and ssa1ts cells, fractionated on a Superose 6 column, and analyzed by immunoblot analysis as described for Fig. 2 except that expression was carried out at 37°C for 4 h. The elution profiles for VHL and TRiC/CCT are presented. Similar elution profiles (shown for CCT4 wt) were obtained for the CCT4wt and the SSA1wt strains. Void, void volume of the Superose 6 column. (B) Effect of the cct4ts mutation on VHL-chaperone interactions. Lysates from cells expressing VBC at 37°C (CCT4wt, lanes 1 and 3; cct4ts, lanes 2 and 4) were analyzed by immunoprecipitation (Ipp.) with anti-VHL antibodies. Associated chaperones were detected by immunoblot analysis (Imm. Blot). Lanes 1 and 2 show results for the 10% input lysate used in the immunoprecipitations; lanes 3 and 4 show immunoprecipitated material. The top panel shows results for immunoblot detection of CCT1, and the middle panel shows results for immunoblot detection of Ssa1/2p. An immunoblot for VHL is included as an immunoprecipitation control (bottom panel).