Hsp90/Hsp70 chaperone machine regulation of the Saccharomyces MAL-activator as determined in vivo using noninducible and constitutive mutant alleles

Abstract

The Hsp90/Hsp70 chaperone machine is an essential regulator of cell growth and division. It is required for activation of select client proteins, chiefly protein kinases and transcription activators and thus plays a major role in regulating intracellular signaling and gene expression. This report demonstrates, in vivo, the association of the Saccharomyces cerevisiae maltose-responsive transcription activator Mal63 (MAL-activator) with the yeast Hsp70 (Ssa1), Hsp90 (Hsp82), and Hop (Sti1) homologs, using a collection of inducible, constitutive, and noninducible alleles. Each class of mutant activator forms a distinctly different stable multichaperone complex in the absence of maltose. Inducible Mal63p associates with Ssa1, Hsp82, and Sti1 and is released in the presence of maltose. Noninducible mal63 mutant proteins bind to Ssa1 alone and do not stably associate with Hsp82 or Sti1. Constitutive MAL-activators bind well to Hsp82 and poorly to Ssa1 and Sti1, but deletion of STI1 restores Ssa1 binding. Taken together, Mal63p regulation requires the formation of Hsp90/Hsp70 subcomplexes comparable to, yet distinct from those observed with previously characterized Hsp90 clients including glucocorticoid receptor and yeast Hap1p. Thus, comparative studies of different client proteins highlight functional diversity in the operation of the Hsp90/Hsp70 chaperone machine.

Figure 1.—

Myc-tagged Hsp82, Ssa1, and Sti1 do not immunoprecipitate in the absence of HA-tagged Mal63. Strains carrying the genomic Myc-tagged alleles and the parental strains lacking the Myc-tagged HSP82 (strains hsc82Δ and CMY1200), SSA1 (strains JN516 and CMY1300), and STI1 (strains W303 and CMY8015) were transformed with plasmid p416GPD-MAL63/3HA carrying the triple-HA-tagged MAL63 or p416GPD-MAL63 carrying the untagged MAL63. Transformants were grown to midlog at 30° in selective synthetic medium lacking uracil and containing 3% glycerol, 2% lactate. Nondenaturing protein extracts were prepared and incubated with anti-HA bound agarose beads as described in materials and methods. Co-immunoprecipitation (Co-IP) samples and the total cell extracts from which they were prepared were analyzed by Western blotting using anti-HA and anti-Myc antibodies. Experiments were done at least in triplicate and representative blots are shown.

Figure 2.—

Maltase expression is dependent on Ssa1 chaperone. Plasmid series p414GPD-SSA1/6His, p414TEF-SSA1/6His, and pUN30-SSA1/6His, expressing 6His-tagged SSA1/6His from the GPD, TEF, and native SSA1 promoters were used to replace the pGAL1-SSA1 plasmid of strain 5B6 (ssa1∷HIS3 ssa2∷LEU2 SSA3 ssa4∷LYS2 pGAL1-SSA1). This strain series was transformed with plasmid pUN90-MAL63/HA. Transformants were grown at 30° in selective synthetic medium lacking uracil and tryptophan and containing 3% glycerol and 2% lactate (v/v) plus the indicated concentration of maltose. Maltase activity was assayed as described in materials and methods and is expressed as nmoles PNPG (p-nitrophenol-α-glucopyranoside) produced per milligram of protein per minute (top). Assays were carried out on at least three independent transformants. The error bars indicate standard deviation from three independent experiments done in duplicate. Western blot analysis was carried out as described in materials and methods on total cell extracts prepared from transformants grown at the indicated maltose concentration (bottom). Blots were probed with anti-6His antibody and anti-PGK antibody was used as a loading control. Analysis was carried out on three independent transformants; a representative blot is shown.

Figure 3.—

Co-immunoprecipitation of inducible Mal63/3HA MAL-activator protein with Ssa1 and Hsp82. Strains CMY1200 (HSP82/Myc hsc82Δ) (top) and CMY1300 (SSA1/Myc ssa2Δ ssa3Δ ssa4Δ) (bottom) were transformed with the plasmid p416GPD-MAL63/3HA harboring the triple-HA-tagged inducible MAL63 MAL-activator gene. Transformants were grown to midlog at 30° in selective synthetic medium lacking uracil and containing 3% glycerol, 2% lactate, with or without 2% maltose. Nondenaturing protein extracts were prepared as described in materials and methods. Co-immunoprecipitation (Co-IP) samples and the total cell extracts from which they were prepared were analyzed by Western blotting using anti-HA and anti-Myc antibodies. Experiments were done at least in triplicate and representative blots are shown.

Figure 4.—

Co-immunoprecipitation of non-inducible MAL-activator mutant alleles with Ssa1 and Hsp82. Strains CMY1200 (HSP82/Myc hsc82Δ) (top) and CMY1300 (SSA1/Myc ssa2Δ ssa3Δ ssa4Δ) (bottom) were transformed with plasmids p416GPD-mal63-283, p416GPD-mal63-331, p416GPD-mal63-364, p416GPD-mal63-401, p416GPD-mal63-467S9V, and p416GPD-mal63-467A9N harboring the triple-HA-tagged alleles of the noninducible MAL-activator mutant genes (Danzi et al. 2003). Transformants were grown as described in Figure 3. Preparation of nondenaturing extracts, co-immunoprecipitation, and Western blot analysis were carried out as described for Figure 3.

Figure 5.—

Maltase expression in strains carrying constitutive MAL-activators is dependent on Hsp90 but not Hsp70. Strains W303 (HSC82 HSP82) and hsc82Δ cpr7Δ (hsc82Δ HSP82 cpr7Δ) (left) were transformed with plasmids p416GPD-MAL63/3HA, carrying the triple-HA-tagged inducible MAL63 or p416GPD-MAL63/43-C and p416GPD-MAL63(1-283)-T247A, carrying the triple-HA-tagged constitutive MAL-activator mutant genes MAL63/43-C and MAL63(1-283)-T247A, respectively (Gibson et al. 1997; Danzi et al. 2000). The strains expressing SSA1 from the GPD and native SSA1 promoters described above in Figure 2 were transformed with plasmids p416GPD-MAL63/3HA, p416GPD-MAL63/43-C, and p416GPD-MAL63(1-283)-T247A (right). Transformants were grown at 30° in selective synthetic medium lacking the appropriate nutrients for plasmid selection and containing 3% glycerol and 2% lactate (v/v) with 1% maltose added to transformants carrying MAL63/3HA. Maltase activity was assayed on at least three independent transformants as described in Figure 2.

Figure 6.—

Co-immunoprecipitation of constitutive MAL-activator mutant proteins with Ssa1 and Hsp82. Strains CMY1200 (HSP82/Myc hsc82Δ) (top) and CMY1300 (SSA1/Myc ssa2Δ ssa3Δ ssa4Δ) (bottom) were transformed with the plasmids p416GPD-MAL63/43-C and p416GPD-MAL63(1-283)-T247A harboring the triple-HA-tagged constitutive MAL-activator mutant genes MAL63/43-C and MAL63(1-283)-T247A. Transformants were grown as described in Figure 3. Preparation of nondenaturing extracts, co-immunoprecipitation, and Western blot analysis were carried out as described for Figure 3.

Figure 7.—

Maltase expression is dependent on STI1. Strains CMY1200 (STI1 HSP82/Myc hsc82Δ) and CMY8001 (sti1Δ HSP82/Myc hsc82Δ) were transformed with plasmid YCp50-MAL63 or YCp50-MAL43-C carrying the inducible MAL63 and constitutive MAL43-C MAL-activator alleles, respectively. Transformants were grown at 30° in selective synthetic medium lacking uracil and containing 3% glycerol and 2% lactate (v/v) plus the indicated concentration of maltose. Maltase activity was assayed in three independent transformants as described in Figure 2.

Figure 8.—

Effect of sti1Δ on the co-immunoprecipitation of inducible Mal63/3HA MAL-activator with Ssa1 and Hsp82. Strains CMY1200 (STI1 HSP82/Myc hsc82Δ), CMY8001 (sti1Δ HSP82/Myc hsc82Δ), CMY1300 (STI1 SSA1/Myc ssa2Δ ssa3Δ ssa4Δ), and CMY8002 (sti1Δ SSA1/Myc ssa2Δ ssa3Δ ssa4Δ) were transformed with plasmids p416GPD-MAL63/3HA or p416GPD-MAL63/43-C carrying triple-HA-tagged MAL63 and MAL63/43-C, respectively. Transformants were grown as described in Figure 3. Preparation of nondenaturing extracts, co-immunoprecipitation, and Western blot analysis were carried out as described for Figure 3.

Figure 9.—

Co-immunoprecipitation of inducible, constitutive, and noninducible MAL-activators with Sti1. Strain CMY8015 (STI1/Myc) was transformed with plasmids p416GPD-MAL63/3HA, p416GPD-MAL63/43-C, and p416-mal63-401 carrying the triple-HA-tagged alleles of inducible MAL63, constitutive MAL63/43-C, and noninducible mal63-401. Transformants were grown as described in Figure 3. Preparation of nondenaturing extracts, co-immunoprecipitation, and Western blot analysis were carried out as described for Figure 3.

Figure 10.—

Model of MAL-activator regulation by the Hsp90/Hsp70 chaperone cycle.