Effect of mutation of the tetratricopeptide repeat and asparatate-proline 2 domains of Sti1 on Hsp90 signaling and interaction in Saccharomyces cerevisiae

Abstract

Through simultaneous interactions with Hsp70 and Hsp90 via separate tetratricopeptide repeat (TPR) domains, the cochaperone protein Hop/Sti1 has been proposed to play a critical role in the transfer of client proteins from Hsp70 to Hsp90. However, no prior mutational analysis demonstrating a critical in vivo role for the TPR domains of Sti1 has been reported. We used site-directed mutagenesis of the TPR domains combined with a genetic screen to isolate mutations that disrupt Sti1 function. A single amino acid alteration in TPR2A disrupted Hsp90 interaction in vivo but did not significantly affect function. However, deletion of a conserved residue in TPR2A or mutations in the carboxy-terminal DP2 domain completely disrupted Sti1 function. Surprisingly, mutations in TPR1, previously shown to interact with Hsp70, were not sufficient to disrupt in vivo functions unless combined with mutations in TPR2B, suggesting that TPR1 and TPR2B have redundant or overlapping in vivo functions. We further examined the genetic and physical interaction of Sti1 with a mutant form of Hsp90, providing insight into the importance of the TPR2A domain of Sti1 in regulating Hsp90 function.

Figure 1.

Targeted Sti1 mutants. (A) Schematic of the domains of Sti1. The position of targeted conserved basic residues in TPR1 (R79), TPR2A (R341), and TPR2B (R469) are indicated. (B) Growth of sti1 mutants in an sti1 strain. Plasmids expressing WT or mutant STI1 or vector as control (−) were transformed into strain JJ623 (sti1). Transformants were grown overnight and 10-fold serial dilutions were plated on selective media and incubated for 2 days at the indicated temperature. (C) Cell lysates from sti1 disruption strain expressing indicated mutants were analyzed by SDS–PAGE (10% acrylamide) and immunoblotted with a polyclonal antiserum specific for Sti1 or an antibody against the mitochondrial Tim44 protein as a loading control. Lane 1, Wt Sti1; lane 2, vector control; lane 3, R79A; lane 4, R341A; lane 5, R469A; lane 6, R79A + R341A; lane 7, R79A + R469A; lane 8, R341A + R469A; lane 9, R79A + R341A + R469A; lane 10, N520.

Figure 2.

Viablity of sti1 mutants in an sti1ydj1 strain. (A) Strain JJ609 (sti1ydj1/pRS316–YDJ1) was transformed with plasmids containing WT STI1 or sti1 mutants. After overnight incubation at 30°, equal amounts of cells were plated onto selective media (C) to maintain WT YDJ1 or media containing 5-FOA to counterselect for YDJ1 (FOA). Plates were incubated at 25° for 5 days. (B) Genetic screen to isolate sti1 mutants that fail to support viability of a sti1ydj1 strain. A plasmid DNA library containing random mutations in STI1 was transformed into strain JJ609. Transformants were patched onto media to maintain YDJ1 or onto media containing 5-FOA to counterselect for YDJ1. Colonies that failed to grow in the presence of 5-FOA were selected for further study.

Figure 3.

Sti1 mutants obtained in genetic screen. (A) Mutant sti1 plasmids obtained in the genetic screen were transformed into strain JJ623 (sti1). Equal amounts of lysates from sti1 cells expressing WT STI1 or indicated sti1 mutants were separated by SDS–PAGE (10% acrylamide) and immunoblotted with Sti1-specific antibodies. (B) Growth of serial dilutions of the same strains after a 2-day incubation at 37° on selective medium.

Figure 4.

Hsp90 client activity is disrupted by sti1 mutation. (A) Wild-type or sti1 mutant yeast cultures expressing either the GAL1–v-src (pBv-src) multicopy plasmid or the control plasmid (pB656) were grown overnight in selective media containing raffinose as the carbon source. v-src expression was induced by addition of 20% galactose to a final concentration of 2%. Cells were harvested 6 hr after induction. Upper panel shows immunoblot of yeast lysates using anti-phosphotyrosine antibody 4G10 (Upstate Biologicals). Lower panel shows immunoblot of yeast lysates using control antibody against Tim44 as a protein-loading control. (B) GR activity in sti1 mutant strains. sti1 disruption strain JJ623 containing GR (pRS424GPDGR) and corresponding reporter plasmid (pUCΔ55-26X) was transformed with indicated sti1 mutants expressed on low-copy plasmids. Yeast cultures were grown in selective media overnight at 25°, then diluted into fresh media and grown to mid-log phase. β-Galactosidase assays were performed as described in materials and methods.

Figure 5.

Comparison of GR activity in sti1 mutant strains. WT STI1, vector alone (−) and indicated sti1 mutants were transformed into strain JJ623 containing GR (pRS424GPDGR) and reporter plasmid (pUCΔ55-26X). GR activity was measured as in Figure 4 after 1-hr incubation in the presence of 0.1 μM DOC. This data is combined from different experiments in which activity from each experiment is expressed as a percentage of WT activity.

Figure 6.

In vivo interaction of Sti1 with Hsc82/Hsp82. Cell lysates were prepared from strain JJ623 expressing WT and mutant Sti1. Sti1 was immunoprecipitated with a monoclonal antibody against Sti1. Resin-bound complexes were separated by SDS–PAGE and immunoblotted with antibodies specific for Sti1, Hsc82/Hsp82 (Hsp90), or Ssa1/2 (Hsp70).

Figure 7.

Genetic interaction between Sti1 and Hsp82-G313S. (A) Strain JJ816 (hsc82hsp82) expressing hsp82-G313S from a low copy plasmid was transformed with WT or mutant STI1 expressed from a multicopy plasmid. Transformants were grown overnight and 10-fold serial dilutions were plated on selective media and grown for 2 days at the indicated temperature. (B) Strain JJ832 (hsc82hsp82sti1/YEp24–HSP82) expressing hsp82–G313S was transformed with WT or mutant STI1 and plated on 5-FOA. Viable strains were grown overnight, serially diluted, plated on rich medium, and grown for 2 days at the indicated temperature.

Figure 8.

In vivo interaction of Sti1 with Hsp82-G313S. Sti1 was immunoprecipitated from cell lysates as in Figure 6. Lanes 1 and 2, strain JJ816 (hsc82hsp82) expressing WT Hsp82 (lane 1) or Hsp82–G313S (lane 2); lanes 3–6, strain JJ832 (hsc82hsp82sti1); lane 3, WT Hsp82 with no STI1 present; Lanes 4–6, Hsp82–G313S plus WT Sti1 (lane 4), R341A (lane 5), or R341E (lane 6).

Figure 9.

Model for the interaction of Sti1/Hop with Hsp70 and Hsp90. See text for details.