PMC

Precipitation of Cpr7 coprecipitates Cns1-3HA. Wild-type cells harboring the multicopy plasmid pJM111 expressing HA epitope-tagged Cns1 were cultured and lysates prepared as described in Materials and Methods. Cell lysates were incubated with glutathione beads charged with either GST or GST-Cpr7 fusion protein. Proteins precipitated by either GST or GST-Cpr7 or total cellular proteins (Lysate) were identified by immunoblotting with the appropriate antibodies.

TPR motifs in Cns1. The location of each of the three TPR motifs in Cns1 is depicted by a hatched box. The TPR motifs of CN21 are compared with those of Cpr6 and Cpr7. Sites containing identical amino acids are shaded. Underlined amino acids correspond to residues that fit the TPR consensus motif *--*G-*Y/F-----*--A*--Y/F--A*-*-P----- (), where ∗ represents any large hydrophobic amino acid and - represents any amino acid.

Overexpression of CNS1 suppresses the slow-growth phenotype of cpr7Δ hsc82Δ and cpr7Δ cells. cpr7Δ hsc82Δ and cpr7Δ cells harboring multicopy CNS1 plasmids were streaked to selective medium and incubated for 2 days at 30°C. (A) A multicopy plasmid containing the genomic region encompassing CNS1/YBR155w is able to suppress the slow growth of cpr7Δ hsc82Δ cells. Wild-type (HSC82 CPR7) and hsc82Δ cpr7Δ control cells harbor vectors only. (B) Suppression of slow-growth phenotype of cpr7Δ cells by a multicopy CNS1 plasmid (pJM111). Wild-type and cpr7Δ control cells harbor vectors only.

Precipitation of Hsp90 coprecipitates Cns1-3HA. Cells expressing wild-type Hsp90 (Hsp82) or histidine-tagged Hsp90 (Hsp82^FP) were transformed with a low-copy-number (pJM110) or multicopy (pJM111) CNS1::3HA plasmid. Total cellular protein (lanes 1 and 2) and proteins eluted from a nickel affinity matrix (lanes 3 and 4) were analyzed by immunoblotting with the appropriate antibodies. Coprecipitation of Cns1-3HA was also observed from lysates expressing the fusion protein from the low-copy-number plasmid pJM110 (not shown).

Overexpression of CNS1 restores down regulation of HSF activity in cpr7Δ cells. Steady-state β-galactosidase activity in wild-type (CPR7) and cpr7Δ cells harboring the HSF-dependent reporter plasmid pHSE2-lacZ and either vector alone or the multicopy CNS1 plasmid pJM111 (p2μ-CNS1) was measured as described in Materials and Methods. Indicated activities are relative to those of wild-type cells. Each bar represents the mean ± standard deviation of at least six independent experiments.

Overexpression of CNS1 restores GR activity to cpr7Δ cells. cpr7Δ cells harbored a GR-dependent lacZ reporter gene and either the control vector (middle lane) or plasmids expressing CPR7 (pAAD97) or CNS1 (pJM107) as indicated. Cells were treated with hormone and assayed for GR activity as described in Materials and Methods. Each column is the mean ± standard deviation of at least four independent experiments expressed as a percentage of activity of cpr7Δ cells harboring pAAD97.