Domain mapping of Sgt1p sequences required for Skp1p and Ctf13p interaction. (A) In vitro-translated ³⁵S-Sgt1p was incubated with either GST-Skp1p, GST-Ctf13p, or GST immobilized on glutathione Sepharose beads. Bound ³⁵S-Sgt1p was quantified after SDS-PAGE by Phosphorimager analysis. (B) A schematic showing the relative positions of the putative TPR motifs, the HSP20/α-crystallin fold, and the unique, well-conserved region of SGT1 (SGS) is shown. Fragments of Sgt1p were translated in reticulocyte lysates and tested for the ability to bind GST-Skp1p, GST-Ctf13p, or GST as in panel A. The levels of Sgt1p specifically bound to the GST fusion proteins are expressed as a percentage of the input and coded as follows: +++, >25% bound; ++, 11 to 25% bound; +, 5 to 10% bound; −, <5% bound. (C) Temperature-sensitive growth of SGT1 alleles was assessed following 3 days of growth at the indicated temperatures. (D) SGT1 alleles encoding the indicated point mutations were translated and analyzed as in panel A.

CBF3 levels are decreased in sgt1-5 mutant cells. (A) The linear range of the CBF3 band shift assay was confirmed by calculating the relative levels of CBF3-CEN DNA complexes detected with different amounts of extracts added to the reaction mixture from wild-type (⧫) and sgt1-5 mutant (▪) cells grown at room temperature (closed symbol) or 37°C (open symbol) for 3 h. (B) CBF3 levels were measured in the indicated amounts of extracts from wild-type or sgt1-5 mutant cells grown at room temperature or 37°C for 3 h by band shift assay. (C) The average amounts of CBF3-CEN DNA complexes were calculated per microgram of extracts made from SGT1 and sgt1-5 cells grown at 25°C () or 37°C (□). Average values were statistically significantly different (paired Student t test, P < 0.005) between SGT1 and sgt1-5 at both temperatures. (D) Equal amounts of yeast extracts from the indicated strains were supplemented with recombinant Cep3p/Ndc10p or recombinant Ctf13p/Skp1p and analyzed for the CBF3 complex by band shift assay. (E) Relative levels of CBF3 measured when extracts were supplemented with buffer alone (□), recombinant Cep3p/Ndc10 (▪), or recombinant Ctf13p/Skp1p () were quantified as for panel C.

HSP90 regulates the interaction between Skp1p and Sgt1p. (A) Extracts from SGT1, sgt1-3, and sgt1-5 mutant yeast strains grown at 37°C for 3 h were equalized for Sgt1p and immunoprecipitated with antibodies against Sgt1p. Immune complexes were eluted and analyzed by immunoblotting with antibodies against Skp1p or HSP90. The specificity of immunoprecipitates was confirmed with rabbit anti-mouse antibodies (RαM) in place of the Sgt1p antibody. Load represents 2% of the protein used for each IP. (B) Extracts from HSP82, hsp82-T101I, and hsp82-G170D mutant yeast strains grown at 37°C for 3 h were equalized for Sgt1p and analyzed as for panel A. (C) Sgt1-TAP was purified from extracts with IgG Sepharose in the presence of increasing amounts of geldanamycin (GA) and analyzed by immunoblotting with antibodies against Skp1p, Sgt1p, or HSP90. As a control, extract containing untagged Sgt1p was incubated with IgG Sepharose in the presence of dimethyl sulfoxide. (D) Levels of Skp1p and Sgt1p immunoprecipitated in panel C were quantified as described in the legend to Fig. 2F. (E) Purified recombinant MBP-Sgt1p or MBP was prebound to amylose resin and added to extracts from wild-type yeast cells supplemented with the indicated ATP analogue at 5 mM. Following the elution of MBP-Sgt1p, the levels of Skp1p, HSP90, and Sgt1p were determined by immunoblotting with the appropriate antibody. (F) Extracts from SGT1, sgt1-3, and sgt1-5 mutant yeast cells containing the GAL1 promoter upstream of 3XHA-CTF13 and grown at 25 or 37°C in galactose medium for 3 h were immunoprecipitated with antibodies against HA, and immune complexes were eluted and immunoblotted with antibodies against Skp1p and HA to detect 3XHA-Ctf13p. The specificity of immunoprecipitates was confirmed with rabbit anti-mouse antibodies (labeled RαM) in place of the HA antibody. Load represents 2% of the protein used for each IP. (G) Yeast extracts described in panel F were immunoprecipitated with Skp1p antibody, and immune complexes were eluted and immunoblotted against Skp1p and HA to detect 3XHA-Ctf13p.

The carboxy terminus of Sgt1p occludes amino-terminal binding domains. (A) The indicated SGT1 alleles were translated, and their affinity for GST-Skp1p was determined after SDS-PAGE by Phosphorimager analysis. (B) Temperature-sensitive growth of SGT1 alleles was assessed following 3 days of growth on YPD plates at the indicated temperatures. (C) Sgt1p proteolytic fragments were visualized by SDS-PAGE and Coomassie staining following treatment with trypsin for the indicated times (seconds). (D) The levels of full-length Sgt1p (arrow 1 in panel C; ⧫), a fragment representing amino acids 1 to 270 (arrow 2 in panel C; □), and small amino-terminal fragments (arrow 3 in panel C; Δ) were quantified with ImageQuant 5.0 and plotted as a function of time. Protein levels are expressed as a fraction of the input (zero time point). (E) Extracts from SGT1, sgt1-3, and sgt1-5 mutant yeast strains grown at 37°C for 3 h were equalized for Sgt1p and immunoprecipitated with antibodies against Sgt1p. Immune complexes were eluted and analyzed by immunoblotting with Skp1p polyclonal antibodies. The specificity of immunoprecipitates was confirmed with rabbit anti-mouse antibodies (RαM) in place of the Sgt1p antibody. Load represents 2% of the protein used for each IP. (F) Levels of Skp1p that were copurified in panel E were quantified with ImageQuant 5.0, and the data were normalized to the level of Sgt1p immunoprecipitated. (G) Sgt1p was immunoprecipitated from extracts of cells grown to log phase or grown in the presence of α factor (5 μg/ml) or nocodazole (15 μg/ml) for 2 h. Immune complexes were eluted and analyzed by immunoblotting with Skp1p polyclonal antibodies. The specificity of immunoprecipitates was confirmed with preimmune antibodies instead of Sgt1p antibody. Load represents 2% of the protein used for each IP. (H) Yeast cells transformed with a plasmid containing a GAL1 promoter and SGT1 (X), SGT1(1-268) (□), sgt1-5 (Δ), sgt1-5, L31P (○), or the vector alone (+) were grown at 30°C in the presence of galactose. Cell growth was monitored for 9 h, and the number of cells per milliliter was plotted. The lines graphed for the vector alone, SGT1, and sgt1-5, L31P overlap.

Sgt1-5p increases the stability of an Sgt1p-Skp1p-Ctf13p complex. (A) SGT1 and sgt1-5 mutant cells containing the GAL1 promoter upstream of 3XHA-CTF13 were cultured at 37°C in medium containing galactose for 1.5 h, transferred to medium containing dextrose, and maintained at 37°C for 0, 10, 20, 40, 60, 90, 120, and 180 min. Extracts were immunoprecipitated with Sgt1p antibody, and immune complexes were eluted and immunoblotted with antibodies against Skp1p, Sgt1p, and HA to detect 3XHA-Ctf13p. The specificity of immunoprecipitates was confirmed with rabbit preimmune sera in place of the Sgt1p antibody. Load represents 2% of the protein used for each IP. (B) Levels of Skp1p immunoprecipitated with Sgt1p (□) or Sgt1-5p (▪) were quantified as described in the legend to Fig. 2F. To ensure accuracy, IPs were repeated in duplicate. Data presented are the mean and standard deviation calculated from duplicate experiments. Levels of 3XHA-Ctf13p in extract from SGT1 (solid line) or sgt1-5 (dashed line) mutant cells were quantified and normalized to the level in SGT1 mutant cell extract harvested at 0 min. (C) Levels of 3XHA-Ctf13p immunoprecipitated with Sgt1p (□) or Sgt1-5p (▪) depicted in panel A were quantified as described above. Lines represent relative levels of 3XHA-Ctf13p, as described for panel B. (D) CBF3 levels were measured in extracts from SGT1 and sgt1-5 mutant cells by band shift assay and quantified (E) relative to the amount of 3XHA-Ctf13p in SGT1 (□) or sgt1-5 (▪) mutant cell extracts.

HSP90 activity mediates the transient assembly of intermediate CBF3 complexes. HSP90 bound to ATP interacts with Sgt1p and keeps it in a closed conformation, unable to bind to Skp1p and Ctf13p via its amino terminus. Following ATP hydrolysis, HSP90 contributes to the open conformation of Sgt1p, allowing Skp1p and Ctf13p to interact. The nucleotide exchange returns HSP90 to its ATP-bound state and Sgt1p to its closed conformation. The release of Sgt1p-HSP90 allows the final step in CBF3 assembly to occur. The Sgt1-5p mutant is unable to achieve the closed conformation and therefore cannot release Sgt1p-HSP90 from the Ctf13p complex.