Binding of v-Src to the NH₂-terminal half of Cdc37 and construction of yeast Cdc37 truncation mutants. (A) Schematic of human Cdc37 and the Cdc37^1–173 truncation mutant. The Hsp90-binding site is shown as a white box. (B) Binding of v-Src and Hsp90 to human His₆Cdc37 and His₆Cdc37^1–173. V-Src protein was translated in rabbit reticulocyte lysates in the presence of [³⁵S]methionine and incubated with exogenous recombinant His₆Cdc37 and His₆Cdc37^1–173. Complexes were isolated on Ni-NTA resin and eluted with imidazole. Presence of Hsp90 in the eluates was determined by Western blot analysis (top). Presence of v-Src was determined by fluorography (bottom). Lane 1, input (1% of the binding reaction); lane 2, binding reaction with resin only; lane 3, binding reaction with exogenous full-length Cdc37; lane 4, binding reaction with His₆Cdc37^1–173. (C) Schematic of yeast Cdc37 and truncation mutants used in this study. The putative Hsp90-binding site is shown as a white box (between amino acids 243 and 343) and is based on sequence similarity with human Cdc37. The limits for the Hsp90-binding site were deduced from previous studies (Grammatikakis et al., 1999; Scholz et al., 2001). All truncations were synthesized with an NH₂-terminal 6-histidine tag. The ability of each truncation to be expressed in yeast (as determined by Western blot) is noted. (D) Western blot analysis of His₆yCdc37 and the truncation mutants with anti-His₆ after affinity chromatography with Ni-NTA resin. The bands labeled with an asterisk at 86 kD, and ∼50 kD are nonspecific. Molecular size standards are shown at left.

Effect of Cdc37 overexpression on v-Src activity in an hsc82Δ strain and GR in the sti1Δ strain. (A) Western blot analysis of tyrosine kinase activity (P-Tyr) by v-Src in hsc82Δ yeast overexpressing wild-type (lane 3) or truncated forms of CDC37 (lanes 4–7) as indicated. V-Src–dependent tyrosine kinase activity in wild-type cells and hsc82Δ cells with empty vector is shown in lanes 1 and 2. Middle and bottom panels show levels of v-Src and phosphoglycerate kinase (PGK) proteins in the same extracts. Cells were grown at 30°C. (B) CDC37 overexpression does not suppress loss of GR activity in sti1Δ cells. Wild-type (WT) and sti1Δ cells expressing GR were grown to mid-log phase and GR-dependent β-galactosidase activity was induced by addition of 1μM deoxycorticosterone. Gray bars represent basal β-galactosidase activity in wild-type or sti1Δ cells; black bars represent GR-dependent β-galactosidase activity in WT cells; hatched bars represent β-galactosidase activity in sti1Δ cells. The absence (−) or presence (+) of overexpressed CDC37 is noted. Fold induction (ratio of induced β-galactosidase levels divided by basal levels) is shown above bars. Data shown are the mean of three independent experiments ± standard error.

Protein kinase activity in cdc37Δ cells expressing Cdc37^1–148 protein. (A) Phosphotyrosine activity in cells expressing v-Src. Lane 1, wild-type cells; lane 2, cdc37Δ cells expressing full-length Cdc37; lane 3, cdc37Δ cells expressing Cdc37^1–148. Western blot with anti-phosphotyrosine (top), Western blot with anti–v-Src (middle), and Western blot with anti-phosphoglycerate kinase (PGK) (bottom). (B) β-galactosidase activity as a measure of MAP kinase signaling. Cells treated with or without α-factor were assayed for Ste12-dependent lacZ gene expression. All strains were cdc37Δ expressing either cdc37–34, full-length Cdc37, or Cdc37^1–148 proteins as indicated.

Cdc37 overexpression suppresses v-Src activity defect in sti1Δ yeast. (A) Western blot analysis using antiphosphotyrosine (top), anti–v-Src (middle), and antiphosphoglycerate kinase (bottom, Pak). Extracts were prepared after induction of v-Src for 6 h from wild type (lane 1) and sti1Δ strains minus and plus Cdc37 overexpression (lanes 2 and 3). Cells were grown at 25°C. (B) Extracts were prepared as describd for A, except that cells were grown at 30°C. (C) Pull-down assay with GST-Hsp90. V-Src was induced by growth of wild-type (WT) and sti1Δ cells in galactose-containing media. Lane 1, wild-type cells not expressing GST-hsp90; lane 2, wild-type cells expressing GST-Hsp90; lane 3, wild-type cells expressing GST-Hsp90 plus overexpressed Cdc37; lane 4, sti1Δ cells expressing GST-Hsp90; lane 5, sti1Δ cells expressing GST-Hsp90 plus overexpressed Cdc37. The bottom two panels show Western blots of proteins isolated on GST-agarose resin. The top two panels show Western blots of proteins in cell extracts.

Effect of different Cdc37 truncation mutants on v-Src activity in a sti1Δ strain. (A) Western blot analysis of tyrosine kinase activity (P-Tyr) by v-Src in sti1Δ yeast overexpressing wild-type (lane 3) or truncated forms of CDC37 (lanes 4–7) as indicated. V-Src–dependent tyrosine kinase activity in wild-type cells and sti1Δ cells with empty vector is shown in lanes 1 and 2. Middle and bottom panels show levels of v-Src and phosphoglycerate kinase (PGK) proteins in the same extracts. Cells were grown at 25°C. (B) Extracts were prepared as described for A, except that the cells were grown at 30°C.

The protein kinase–binding domain of Cdc37 is sufficient for viability. (A) Spot test analysis of cdc37Δ cells expressing wild-type (Cdc37) or truncated versions of Cdc37 as indicated in the figure. 10-fold dilutions of saturated cultures were plated (3 μl each) and incubated at 30°C and 37°C. (B) Western blot analysis of proteins eluted from Ni-NTA resin after incubation with extracts expressing vector alone (lane 1 or full-length histidine-tagged Cdc37 on a multicopy plasmid (lanes 2–5). Cdc37^1–239 was coexpressed with full-length Cdc37 on low copy number (lane 2) or high copy number plasmids (lane 4). Cdc37^1–148 was similarly expressed from low copy number or high copy number plasmids (lanes 3 and 5, respectively). Proteins that reacted with anti-His6 that bound nonspecifically to the resin are labeled with an asterisk. (C) Growth of cells expressing different forms of CDC37 from low copy or high copy number plasmids as indicated. Cells are shown spotted onto plates the absence or presence of 5-fluoroorotic acid (5-FOA) to counterselect for full-length Cdc37. (D) Spot test analysis of growth of cdc37–34 mutant yeast expressing truncated versions of Cdc37 as indicated. The cells were grown at 25°C or 37°C.

Model for v-Src folding by molecular chaperones. Nascent v-Src is shown to interact first with Hsp70/Hsp40 as do other nascent chains. Hsp90 and Cdc37 recruitment follows interaction of Hop/Sti1 with Hsp70. In yeast mutants deleted for STI1 or having reduced amounts of Hsp90 (in the hsc82Δ strain), a bypass pathway can promote v-Src folding if sufficient Cdc37 is available (dotted line). Our results suggest that the bypass may occur in an Hsp90-independent manner, although Hsp90 makes the reaction more efficient. See text for further details.