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In vivo function of Hsp90 is dependent on ATP binding and ATP hydrolysis.

Obermann WM, Sondermann H, Russo AA, Pavletich NP, Hartl FU - J. Cell Biol. (1998)

Bottom Line: Our results show that both ATP binding and hydrolysis are required for Hsp82 function in vivo.Remarkably, the complete Hsp90 protein is required for ATPase activity and for the interaction with p23, suggesting an intricate allosteric communication between the domains of the Hsp90 dimer.Our results establish Hsp90 as an ATP-dependent chaperone.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Biochemistry, Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany.

ABSTRACT
Heat shock protein 90 (Hsp90), an abundant molecular chaperone in the eukaryotic cytosol, is involved in the folding of a set of cell regulatory proteins and in the re-folding of stress-denatured polypeptides. The basic mechanism of action of Hsp90 is not yet understood. In particular, it has been debated whether Hsp90 function is ATP dependent. A recent crystal structure of the NH2-terminal domain of yeast Hsp90 established the presence of a conserved nucleotide binding site that is identical with the binding site of geldanamycin, a specific inhibitor of Hsp90. The functional significance of nucleotide binding by Hsp90 has remained unclear. Here we present evidence for a slow but clearly detectable ATPase activity in purified Hsp90. Based on a new crystal structure of the NH2-terminal domain of human Hsp90 with bound ADP-Mg and on the structural homology of this domain with the ATPase domain of Escherichia coli DNA gyrase, the residues of Hsp90 critical in ATP binding (D93) and ATP hydrolysis (E47) were identified. The corresponding mutations were made in the yeast Hsp90 homologue, Hsp82, and tested for their ability to functionally replace wild-type Hsp82. Our results show that both ATP binding and hydrolysis are required for Hsp82 function in vivo. The mutant Hsp90 proteins tested are defective in the binding and ATP hydrolysis-dependent cycling of the co-chaperone p23, which is thought to regulate the binding and release of substrate polypeptide from Hsp90. Remarkably, the complete Hsp90 protein is required for ATPase activity and for the interaction with p23, suggesting an intricate allosteric communication between the domains of the Hsp90 dimer. Our results establish Hsp90 as an ATP-dependent chaperone.

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Mutations in the nucleotide-binding site of Hsp90 abolish the function of Hsp90 in vivo. (A) Yeast strain ΔPCLDa/α  that expresses wild-type Hsc82 from the plasmid pKAT6 (containing the URA3-marker) was cotransformed with wild-type  HSP82 (lane 1), HSP82His6EEF (lane 2), HSP82(E33A)His6EEF  (lane 3), HSP82(E33D)His6EEF (lane 4) and HSP82(D79N)  His6EEF (lane 5). After growth to mid-log phase in liquid SD/ −Trp/−Ura, cell lysates were prepared, adjusted to equal protein  concentrations and His6EEF tagged proteins quantitatively absorbed with Ni-NTA beads followed by SDS-PAGE and Coomassie staining (lanes 1–5). Lanes 6–10 correspond to lanes 1–5  and show an immunoblot with the EEF-specific antibody. Note  that only the band at ∼90 kD (and two degradation products at  ∼60 and ∼50 kD, generated during isolation) are specifically recognized. (B) The cotransformants described in A were restreaked  on 5-FOA plates at 25°C to select for cells that had lost the original wild-type plasmid and were rescued from lethality by a functional hsp82 protein. Only wild-type Hsp82, Hsp82His6EEF, and  the E33D mutant were able to support growth.
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Figure 2: Mutations in the nucleotide-binding site of Hsp90 abolish the function of Hsp90 in vivo. (A) Yeast strain ΔPCLDa/α that expresses wild-type Hsc82 from the plasmid pKAT6 (containing the URA3-marker) was cotransformed with wild-type HSP82 (lane 1), HSP82His6EEF (lane 2), HSP82(E33A)His6EEF (lane 3), HSP82(E33D)His6EEF (lane 4) and HSP82(D79N) His6EEF (lane 5). After growth to mid-log phase in liquid SD/ −Trp/−Ura, cell lysates were prepared, adjusted to equal protein concentrations and His6EEF tagged proteins quantitatively absorbed with Ni-NTA beads followed by SDS-PAGE and Coomassie staining (lanes 1–5). Lanes 6–10 correspond to lanes 1–5 and show an immunoblot with the EEF-specific antibody. Note that only the band at ∼90 kD (and two degradation products at ∼60 and ∼50 kD, generated during isolation) are specifically recognized. (B) The cotransformants described in A were restreaked on 5-FOA plates at 25°C to select for cells that had lost the original wild-type plasmid and were rescued from lethality by a functional hsp82 protein. Only wild-type Hsp82, Hsp82His6EEF, and the E33D mutant were able to support growth.

Mentions: To test the possible functional significance of the conserved D93 and E47 residues of human Hsp90 for ATP binding and hydrolysis, respectively, the effects of site-directed mutations in the corresponding residues D79 and E33 of yeast Hsp82 (83% similarity and 70% identity with the human sequence) were analyzed in vivo. Yeast contains two HSP90 genes, HSP82 and HSC82, whose combined deletion is lethal (Borkovich et al., 1989). We therefore used a yeast strain (ΔPCLDa/α) for this analysis that is deleted in both chromosomal HSP90 genes but rescued from lethality by constitutive expression of plasmid-encoded wild-type Hsc82 protein (Nathan and Lindquist, 1995). We first addressed the question whether mutations of D79 and E33 affect the expression and gross stability of Hsp82. The ΔPCLDa/α strain was cotransformed with an additional HSP82 gene expressing either wild-type or mutant proteins (from the same promoter) that could be distinguished from the preexistent Hsc82 by the presence of COOH-terminal His6 and EEF tags (Wehland et al., 1984). Yeast cells expressing either untagged wild-type Hsp82, wild-type Hsp82-His6EEF, or Hsp82-His6EEF proteins containing the mutations E33A, E33D, or D79N were grown to mid-log phase. Cell lysates were prepared, adjusted to equal protein concentrations, and then Hsp82 proteins quantitatively adsorbed to Ni-NTA beads via their His6 tags (see Materials and Methods). All Hsp82-His6EEF proteins were stably expressed as full-length proteins to very similar levels (Fig. 2 A), as judged by Coomassie staining and immunoblotting with anti-EEF antibody. The additional bands at ∼50 and ∼60 kD in the Western blot represent degradation products of the full-length protein that are generated during the isolation procedure, as they are not detected on Western blots of cells that had been solubilized in hot SDS immediately upon harvesting (not shown). Coexpression of mutant proteins at a level equivalent to that of wild-type Hsp82 was without effect on cell growth.


In vivo function of Hsp90 is dependent on ATP binding and ATP hydrolysis.

Obermann WM, Sondermann H, Russo AA, Pavletich NP, Hartl FU - J. Cell Biol. (1998)

Mutations in the nucleotide-binding site of Hsp90 abolish the function of Hsp90 in vivo. (A) Yeast strain ΔPCLDa/α  that expresses wild-type Hsc82 from the plasmid pKAT6 (containing the URA3-marker) was cotransformed with wild-type  HSP82 (lane 1), HSP82His6EEF (lane 2), HSP82(E33A)His6EEF  (lane 3), HSP82(E33D)His6EEF (lane 4) and HSP82(D79N)  His6EEF (lane 5). After growth to mid-log phase in liquid SD/ −Trp/−Ura, cell lysates were prepared, adjusted to equal protein  concentrations and His6EEF tagged proteins quantitatively absorbed with Ni-NTA beads followed by SDS-PAGE and Coomassie staining (lanes 1–5). Lanes 6–10 correspond to lanes 1–5  and show an immunoblot with the EEF-specific antibody. Note  that only the band at ∼90 kD (and two degradation products at  ∼60 and ∼50 kD, generated during isolation) are specifically recognized. (B) The cotransformants described in A were restreaked  on 5-FOA plates at 25°C to select for cells that had lost the original wild-type plasmid and were rescued from lethality by a functional hsp82 protein. Only wild-type Hsp82, Hsp82His6EEF, and  the E33D mutant were able to support growth.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132952&req=5

Figure 2: Mutations in the nucleotide-binding site of Hsp90 abolish the function of Hsp90 in vivo. (A) Yeast strain ΔPCLDa/α that expresses wild-type Hsc82 from the plasmid pKAT6 (containing the URA3-marker) was cotransformed with wild-type HSP82 (lane 1), HSP82His6EEF (lane 2), HSP82(E33A)His6EEF (lane 3), HSP82(E33D)His6EEF (lane 4) and HSP82(D79N) His6EEF (lane 5). After growth to mid-log phase in liquid SD/ −Trp/−Ura, cell lysates were prepared, adjusted to equal protein concentrations and His6EEF tagged proteins quantitatively absorbed with Ni-NTA beads followed by SDS-PAGE and Coomassie staining (lanes 1–5). Lanes 6–10 correspond to lanes 1–5 and show an immunoblot with the EEF-specific antibody. Note that only the band at ∼90 kD (and two degradation products at ∼60 and ∼50 kD, generated during isolation) are specifically recognized. (B) The cotransformants described in A were restreaked on 5-FOA plates at 25°C to select for cells that had lost the original wild-type plasmid and were rescued from lethality by a functional hsp82 protein. Only wild-type Hsp82, Hsp82His6EEF, and the E33D mutant were able to support growth.
Mentions: To test the possible functional significance of the conserved D93 and E47 residues of human Hsp90 for ATP binding and hydrolysis, respectively, the effects of site-directed mutations in the corresponding residues D79 and E33 of yeast Hsp82 (83% similarity and 70% identity with the human sequence) were analyzed in vivo. Yeast contains two HSP90 genes, HSP82 and HSC82, whose combined deletion is lethal (Borkovich et al., 1989). We therefore used a yeast strain (ΔPCLDa/α) for this analysis that is deleted in both chromosomal HSP90 genes but rescued from lethality by constitutive expression of plasmid-encoded wild-type Hsc82 protein (Nathan and Lindquist, 1995). We first addressed the question whether mutations of D79 and E33 affect the expression and gross stability of Hsp82. The ΔPCLDa/α strain was cotransformed with an additional HSP82 gene expressing either wild-type or mutant proteins (from the same promoter) that could be distinguished from the preexistent Hsc82 by the presence of COOH-terminal His6 and EEF tags (Wehland et al., 1984). Yeast cells expressing either untagged wild-type Hsp82, wild-type Hsp82-His6EEF, or Hsp82-His6EEF proteins containing the mutations E33A, E33D, or D79N were grown to mid-log phase. Cell lysates were prepared, adjusted to equal protein concentrations, and then Hsp82 proteins quantitatively adsorbed to Ni-NTA beads via their His6 tags (see Materials and Methods). All Hsp82-His6EEF proteins were stably expressed as full-length proteins to very similar levels (Fig. 2 A), as judged by Coomassie staining and immunoblotting with anti-EEF antibody. The additional bands at ∼50 and ∼60 kD in the Western blot represent degradation products of the full-length protein that are generated during the isolation procedure, as they are not detected on Western blots of cells that had been solubilized in hot SDS immediately upon harvesting (not shown). Coexpression of mutant proteins at a level equivalent to that of wild-type Hsp82 was without effect on cell growth.

Bottom Line: Our results show that both ATP binding and hydrolysis are required for Hsp82 function in vivo.Remarkably, the complete Hsp90 protein is required for ATPase activity and for the interaction with p23, suggesting an intricate allosteric communication between the domains of the Hsp90 dimer.Our results establish Hsp90 as an ATP-dependent chaperone.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Biochemistry, Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany.

ABSTRACT
Heat shock protein 90 (Hsp90), an abundant molecular chaperone in the eukaryotic cytosol, is involved in the folding of a set of cell regulatory proteins and in the re-folding of stress-denatured polypeptides. The basic mechanism of action of Hsp90 is not yet understood. In particular, it has been debated whether Hsp90 function is ATP dependent. A recent crystal structure of the NH2-terminal domain of yeast Hsp90 established the presence of a conserved nucleotide binding site that is identical with the binding site of geldanamycin, a specific inhibitor of Hsp90. The functional significance of nucleotide binding by Hsp90 has remained unclear. Here we present evidence for a slow but clearly detectable ATPase activity in purified Hsp90. Based on a new crystal structure of the NH2-terminal domain of human Hsp90 with bound ADP-Mg and on the structural homology of this domain with the ATPase domain of Escherichia coli DNA gyrase, the residues of Hsp90 critical in ATP binding (D93) and ATP hydrolysis (E47) were identified. The corresponding mutations were made in the yeast Hsp90 homologue, Hsp82, and tested for their ability to functionally replace wild-type Hsp82. Our results show that both ATP binding and hydrolysis are required for Hsp82 function in vivo. The mutant Hsp90 proteins tested are defective in the binding and ATP hydrolysis-dependent cycling of the co-chaperone p23, which is thought to regulate the binding and release of substrate polypeptide from Hsp90. Remarkably, the complete Hsp90 protein is required for ATPase activity and for the interaction with p23, suggesting an intricate allosteric communication between the domains of the Hsp90 dimer. Our results establish Hsp90 as an ATP-dependent chaperone.

Show MeSH
Related in: MedlinePlus