Hsp90 Mutants Research Articles

Role for Hsp70 Chaperone in Saccharomyces cerevisiae Prion Seed Replication

The Saccharomyces cerevisiae [PSI+] prion is a misfolded form of Sup35p that propagates as self-replicating cytoplasmic aggregates. Replication is believed to occur through breakage of transmissible [PSI+] prion particles, or seeds, into more numerous pieces. In [PSI+] cells, large Sup35p aggregates are formed by coalescence of smaller sodium dodecyl sulfate-insoluble polymers. It is uncertain if polymers or higher-order aggregates or both act as prion seeds. A mutant Hsp70 chaperone, Ssa1-21p, reduces the number of transmissible [PSI+] seeds per cell by 10-fold but the overall amount of aggregated Sup35p by only two- to threefold. This discrepancy could be explained if, in SSA1-21 cells, [PSI+] seeds are larger or more of the aggregated Sup35p does not function as a seed. To visualize differences in aggregate size, we constructed a Sup35-green fluorescent protein (GFP) fusion (NGMC) that has normal Sup35p function and can propagate like [PSI+]. Unlike GFP fusions lacking Sup35p's essential C-terminal domain, NGMC did not form fluorescent foci in log-phase [PSI+] cells. However, using fluorescence recovery after photobleaching and size fractionation techniques, we find evidence that NGMC is aggregated in these cells. Furthermore, the aggregates were larger in SSA1-21 cells, but the size of NGMC polymers was unchanged. Possibly, NGMC aggregates are bigger in SSA1-21 cells because they contain more polymers. Our data suggest that Ssa1-21p interferes with disruption of large Sup35p aggregates, which lack or have limited capacity to function as seed, into polymers that function more efficiently as [PSI+] seeds.

Co-chaperone Regulation of Conformational Switching in the Hsp90 ATPase Cycle

ATP hydrolysis by the Hsp90 molecular chaperone requires a connected set of conformational switches triggered by ATP binding to the N-terminal domain in the Hsp90 dimer. Central to this is a segment of the structure, which closes like a "lid" over bound ATP, promoting N-terminal dimerization and assembly of a competent active site. Hsp90 mutants that influence these conformational switches have strong effects on ATPase activity. ATPase activity is specifically regulated by Hsp90 co-chaperones, which directly influence the conformational switches. Here we have analyzed the effect of Hsp90 mutations on binding (using isothermal titration calorimetry and difference circular dichroism) and ATPase regulation by the co-chaperones Aha1, Sti1 (Hop), and Sba1 (p23). The ability of Sti1 to bind Hsp90 and arrest its ATPase activity was not affected by any of the mutants screened. Sba1 bound in the presence of AMPPNP to wild-type and ATPase hyperactive mutants with similar affinity but only very weakly to hypoactive mutants despite their wild-type ATP affinity. Unexpectedly, in all cases Sba1 bound to Hsp90 with a 1:2 molar stoichiometry. Aha1 binding to mutants was similar to wild-type, but the -fold activation of their ATPase varied substantially between mutants. Analysis of complex formation with co-chaperone mixtures showed Aha1 and p50cdc37 able to bind Hsp90 simultaneously but without direct interaction. Sba1 and p50cdc37 bound independently to Hsp90-AMPPNP but not together. These data indicated that Sba1 and Aha1 regulate Hsp90 by influencing the conformational state of the "ATP lid" and consequent N-terminal dimerization, whereas Sti1 does not.

Overexpression of Wild-Type Heat Shock Protein 27 and a Nonphosphorylatable Heat Shock Protein 27 Mutant Protects Against Ischemia/Reperfusion Injury in a Transgenic Mouse Model

The small heat shock protein 27 (hsp27) increases in expression with ischemia/reperfusion (I/R) insult in the heart. One feature of the small hsps is their ability to oligomerize and form intracellular aggregates. Oligomerization pattern is governed by the phosphorylation state of the protein that may influence their ability to protect against cellular stresses. We generated transgenic (tg) mice that overexpress a wild-type human hsp27 (hsp27tg) protein or a mutant hsp27 protein (mut-hsp27tg), in which serine residues (aa15, aa78, and aa82) were replaced by alanine residues, rendering them incapable of phosphorylation. Using a Langendorff perfusion model and an intraventricular balloon, we subjected hearts to 20 minutes of ischemia followed by 1 hour of reperfusion. During reperfusion, negative and positive pressure derivatives as well as developed pressures were significantly higher in both hsp27tg and mut-hsp27tg compared with control (P<0.01) mice, with no significant difference between hsp27tg and mut-hsp27tg. Creatine kinase release during reperfusion was higher in control compared with both hsp27tg and mut-hsp27tg (P<0.05). Malondialdehyde content as well as protein oxidation products were lower in mut-hsp27tg compared with control (P<0.05). hsp27tg hearts possessed oligomers that ranged in size from small to large, whereas mut-hsp27tg hearts contained no small oligomers. These results indicate that in a tg mouse model, overexpression of either wild-type hsp27 or a nonphosphorylatable hsp27 mutant was equally capable of protecting the heart from I/R injury. Furthermore, the phosphorylation status of hsp27 may influence its ability to decrease oxidative stress.

Proteasome inhibitors suppress formation of polyglutamine‐induced nuclear inclusions in cultured postmitotic neurons

At least nine neurodegenerative disorders are caused by expansion of polyglutamine repeats in various genes. This expansion induces the formation of nuclear inclusions (NI) within various cell types. In this study, we developed a model for polyglutamine diseases using primary cultures of sympathetic neurons from the superior cervical ganglia of prenatal rat pups. Transfection with a plasmid encoding 127 glutamine repeats causes NI to develop in approximately 70% of the sympathetic neurons within 6 days. In addition, it causes somatic atrophy and inhibits dendritic growth. The NIs contain ubiquitinated proteins and sequester the molecular chaperone heat shock protein 70 (Hsp70). We found that two specific proteasome inhibitors, lactacystin and CEP1612, suppress thezformation of polyglutamine-induced NI. In addition, lactacystin treatment induced the removal of preexisting NI. Western blotting and immunocytochemistry revealed that lactacystin and CEP1612 strongly induce the expression of Hsp70, whereas less specific proteasome inhibitor such as N-acetyl-Leu-Leu-Norleucinal does not. Coexpression of 127 glutamines with a plasmid encoding wild-type Hsp70 gene resulted in a marked reduction of the percentage of neurons containing NI. In addition, transfection with plasmids encoding mutant Hsp70 blocked the effects of lactacystin. These findings further implicate Hsp70 as a neuroprotective molecule and they suggest the potential utility of certain proteasome inhibitors in the treatment of polyglutamine diseases.

Hsp70 dynamics in vivo: effect of heat shock and protein aggregation.

The molecular chaperone Hsp70 interacts with misfolded proteins and also accumulates in the nucleus during heat shock. Using GFP-Hsp70 and fluorescence recovery after photobleaching, we show that Hsp70 accumulates in the nucleus during heat shock not only because its inflow rate increases but also because of a marked decrease in its outflow rate. Dynamic imaging also shows that GFP-Hsp70 has greatly reduced mobility when it interacts with organelles such as nucleoli in heat-shocked cells or the large inclusions formed from fragments of mutant huntingtin protein. In heat-shocked cells, nucleoplasmic Hsp70 has reduced mobility relative to the cytoplasm, whereas the ATPase-deficient mutant of Hsp70, Hsp70(K71E), is almost completely immobilized both in the nucleoplasm and the cytoplasm. Moreover, the Hsp70 mutant shows reduced mobility in the presence of diffusive huntingtin fragments with expanded polyglutamine repeats. This provides strong evidence that Hsp70 interacts not only with organelles but also with diffusive proteins in the nucleoplasm and cytoplasm during heat shock as well as with diffusive huntingtin fragments.

Distinct roles for the Hsp40 and Hsp90 molecular chaperones during cystic fibrosis transmembrane conductance regulator degradation in yeast.

Aberrant secreted proteins can be destroyed by ER-associated protein degradation (ERAD), and a prominent, medically relevant ERAD substrate is the cystic fibrosis transmembrane conductance regulator (CFTR). To better define the chaperone requirements during CFTR maturation, the protein was expressed in yeast. Because Hsp70 function impacts CFTR biogenesis in yeast and mammals, we first sought ER-associated Hsp40 cochaperones involved in CFTR maturation. Ydj1p and Hlj1p enhanced Hsp70 ATP hydrolysis but CFTR degradation was slowed only in yeast mutated for both YDJ1 and HLJ1, suggesting functional redundancy. In contrast, CFTR degradation was accelerated in an Hsp90 mutant strain, suggesting that Hsp90 preserves CFTR in a folded state, and consistent with this hypothesis, Hsp90 maintained the solubility of an aggregation-prone domain (NBD1) in CFTR. Soluble ERAD substrate degradation was unaffected in the Hsp90 or the Ydj1p/Hlj1p mutants, and surprisingly CFTR degradation was unaffected in yeast mutated for Hsp90 cochaperones. These results indicate that Hsp90, but not the Hsp90 complex, maintains CFTR structural integrity, whereas Ydj1p/Hlj1p catalyze CFTR degradation.

Peptides complexed with the protein HSP70 generate efficient human cytolytic T-lymphocyte responses

Microbial HSPs (heat-shock proteins) are implicated in the induction of the innate and adaptive arms of the immune response. We set out to determine whether peptides complexed with HSP70 generate efficient CTL (cytolytic T-lymphocyte) responses. Human dendritic cells pulsed with peptide-loaded microbial HSP70 complexes generate potent antigen-specific CTL responses. Using fluorescence anisotropy, we have calculated the peptide-binding affinity of mycobacterial HSP70 (K(D)=14 microM) and show that 120 pM HSP70-bound peptide is sufficient to generate a peptide-specific CTL response that is four orders of magnitude more efficient than the peptide alone. Through the generation of mycobacterial HSP70 truncations, we find that the minimal 136 amino acid, mycobacterial HSP70 peptide-binding domain is sufficient to generate CTL responses. The design of an HSP70 mutant, in which the peptide-binding site of HSP70 is filled with a bulky hydrophobic residue, leads to a large decrease in the peptide-binding affinity. This mutant HSP70 retains stimulatory capacity but is unable to generate CTL and has separated antigen delivery from immunostimulation of dendritic cells.

Multiple Domains of the Co-chaperone Hop Are Important for Hsp70 Binding

The Hop/Sti1 co-chaperone binds to both Hsp70 and Hsp90. Biochemical and co-crystallographic studies have suggested that the EEVD-containing C terminus of Hsp70 or Hsp90 binds specifically to one of the Hop tetratricopeptide repeat domains, TPR1 or TPR2a, respectively. Mutational analyses of Hsp70 and Hop were undertaken to better characterize interactions between the C terminus of Hsp70 and Hop domains. Surprisingly, truncation of EEVD plus as many as 34 additional amino acids from the Hsp70 C terminus did not reduce the ability of Hsp70 mutants to co-immunoprecipitate with Hop, although further truncation eliminated Hop binding. Hop point mutations targeting a carboxylate clamp position in TPR1 disrupted Hsp70 binding, as was expected; however, similar point mutations in TPR2a or TPR2b also inhibited Hsp70 binding in some settings. Using a yeast-based in vivo assay for Hop function, wild type Hop and TPR2b mutants could fully complement deletion of Sti1p; TPR1 and TPR2a point mutants could partially restore activity. Conformations of Hop and Hop mutants were probed by limited proteolysis. The TPR1 mutant digested in a similar manner to wild type; however, TPR2a and TPR2b mutants each displayed greater resistance to chymotryptic digestion. All point mutants retained an ability to dimerize, and none appeared to be grossly misfolded. These results raise questions about current models for Hop/Hsp70 interaction.

Cooperation of heat shock protein 90 and p23 in aryl hydrocarbon receptor signaling.

Aryl hydrocarbon receptor (AhR) is a transcription factor that is activated by the binding of xenobiotic and endogenous ligands. AhR interacts with heat shock protein (Hsp) 90 complexes and can be used as a functional substrate to detect chaperone-dependent processes. Yeast Hsp90 (hsp82) mutants that variably affected AhR signaling were identified using reporter gene assays. Some mutated alleles resided in the p23/adenosine triphosphate (ATP)-binding pocket of Hsp90, so the relationship between the cochaperone Sba1 (yeast p23) and adenosine triphosphatase (ATPase) activity was investigated. Deletion of the p23 gene in the hsp82G170D mutant background had a greater effect on AhR signaling than the individual mutations, suggesting that these 2 mutations have separate actions on AhR signaling. In contrast, p23 overexpression suppressed temperature sensitivity and AhR signaling defects in the hsp82G170D mutant strain, suggesting that there is a relationship between these 2 proteins. The mutated hsp82G170D protein lacked detectable ATPase activity and p23 binding in vitro, which may relate to the weakened AhR signaling observed in mutant cells. Sba1 (p23) suppressed Hsp82 ATPase activity in vitro. These studies implicate the p23 protein and the G170 region of Hsp90 as being important, but not essential, for AhR signaling. Our results are consistent with a model in which p23 inhibits Hsp90 ATPase activity, thereby stabilizing ATP-Hsp90-client protein complexes.

Hsp70-DnaJ chaperone pair prevents nitric oxide- and CHOP-induced apoptosis by inhibiting translocation of Bax to mitochondria.

We reported that the endoplasmic reticulum (ER) stress pathway involving CHOP, a member of the C/EBP transcription factor family, plays a key role in nitric oxide (NO)-mediated apoptosis of macrophages and pancreatic beta cells. We also showed that the cytosolic chaperone pair of hsp70 and dj1 (hsp40/hdj-1) or dj2 (HSDJ/hdj-2) prevents NO-mediated apoptosis upstream of cytochrome c release from mitochondria. To analyze roles of the chaperone pair in preventing apoptosis, RAW 264.7 macrophages stably expressing hsp70 and dj1 or dj2 were established. The chaperone pair prevented LPS/IFN-gamma-induced and NO-mediated apoptosis downstream of CHOP induction. hsp70 mutant protein lacking the ATPase domain or the C-terminal EEVD sequence were not effective in preventing CHOP-induced apoptosis. A mutant dj2 lacking the C-terminal prenylation CaaX motif, was also not effective. When wild-type RAW 264.7 cells were treated with LPS/IFN-gamma, NO-mediated apoptosis was induced, and proapoptotic Bcl-2 family protein Bax was translocated from cytosol to mitochondria. This translocation was prevented in cells stably expressing hsp70/dj2, and in CHOP knockout cells. Overexpression of CHOP in wild-type cells also induced translocation of Bax and this translocation was prevented in cells expressing hsp70/dj2. CHOP-induced apoptosis was prevented by Bax knock-down. Coimmunoprecipitation experiments showed that Bax interacts with both hsp70 and dj1/dj2. ATPase domain of hsp70 was necessary for the binding with Bax. These findings indicate that CHOP-induced apoptosis is mediated by translocation of Bax from the cytosol to the mitochondria, and hsp70/dj1 or dj2 chaperone pair prevents apoptosis by interacting with Bax and preventing translocation to the mitochondria.

Hsp70 mutant proteins modulate additional apoptotic pathways and improve cell survival

Although wild-type Hsp70 (Hsp70WT) inhibits procaspase-3 processing by preventing apoptosome formation, Hsp70WT does not block active caspase-3. Because all caspase-3 inhibitors bear canonical DXXD caspase-3 recognition motifs, we determined whether mutated Hsp70s with caspase-binding motifs act as direct caspase-3 inhibitors. Based on Hsp70 molecular modeling, the DNQP, DEVQ, and EEVD regions localized on the surface of Hsp70WT were chosen, allowing us to design mutants while trying to avoid disrupting the global fold of the molecule and losing the possibility of protein-protein interactions. We replaced DNQP with DQMD, and DEVQ and EEVD with DEVD residues that should be optimal substrates for caspase-3. The resultant Hsp70 mutants directly interacted with active caspase-3 and blocked its proteolytic activity while retaining the ability to reverse protein denaturation and disrupt the interaction between Apaf-1 and procaspase-9. The Hsp70C-terminal mutants interacted with Apaf-1 and active caspase-3 significantly longer than Hsp70WT. The Hsp70 DXXD mutants protected neuron and teratocarcinoma (NT) cells against cell death much better than Hsp70WT whether given before or after serum withdrawal. Hsp70 mutants represent a possible approach to antiapoptotic biotherapeutics. Similar rational designs could be used to engineer inhibitors of additional caspase family members.

Cooperation of heat shock protein 90 and p23 in aryl hydrocarbon receptor signaling

Aryl hydrocarbon receptor (AhR) is a transcription factor that is activated by the binding of xenobiotic and endogenous ligands. AhR interacts with heat shock protein (Hsp) 90 complexes and can be used as a functional substrate to detect chaperone-dependent processes. Yeast Hsp90 (hsp82) mutants that variably affected AhR signaling were identified using reporter gene assays. Some mutated alleles resided in the p23/adenosine triphosphate (ATP)–binding pocket of Hsp90, so the relationship between the cochaperone Sba1 (yeast p23) and adenosine triphosphatase (ATPase) activity was investigated. Deletion of the p23 gene in the hsp82G170D mutant background had a greater effect on AhR signaling than the individual mutations, suggesting that these 2 mutations have separate actions on AhR signaling. In contrast, p23 overexpression suppressed temperature sensitivity and AhR signaling defects in the hsp82G170D mutant strain, suggesting that there is a relationship between these 2 proteins. The mutated hsp82G170D protein lacked detectable ATPase activity and p23 binding in vitro, which may relate to the weakened AhR signaling observed in mutant cells. Sba1 (p23) suppressed Hsp82 ATPase activity in vitro. These studies implicate the p23 protein and the G170 region of Hsp90 as being important, but not essential, for AhR signaling. Our results are consistent with a model in which p23 inhibits Hsp90 ATPase activity, thereby stabilizing ATP-Hsp90-client protein complexes.

HSP70 Peptide Binding Mutants Separate Antigen Delivery from Dendritic Cell Stimulation

Microbial heat shock proteins (HSPs) have been implicated in the induction of both the innate and adaptive arms of the immune response. We now show that human dendritic cells (DC) pulsed with peptide-loaded mycobacterial HSP70 complexes generate potent antigen-specific cytotoxic lymphocyte (CTL) responses, which are dependent on an HSP70-stimulated calcium signaling cascade. From the calculated peptide binding affinity of mycobacterial HSP70 (K D = 14 μM) we show that 120 pM HSP70 bound peptide is sufficient to generate a peptide-specific CTL response that is up to four orders of magnitude more efficient than peptide alone. The minimal 136 amino acid, mycobacterial HSP70 peptide binding domain can generate CTL responses, and a single amino acid mutant HSP70 designed to prevent peptide binding but retain stimulatory capacity has allowed us to separate antigen delivery from DC immunostimulation.

Saccharomyces cerevisiae Hsp70 mutations affect [PSI+] prion propagation and cell growth differently and implicate Hsp40 and tetratricopeptide repeat cochaperones in impairment of [PSI+

We previously described an Hsp70 mutant (Ssa1-21p), altered in a conserved residue (L483W), that dominantly impairs yeast [PSI(+)] prion propagation without affecting growth. We generated new SSA1 mutations that impaired [PSI(+)] propagation and second-site mutations in SSA1-21 that restored normal propagation. Effects of mutations on growth did not correlate with [PSI(+)] phenotype, revealing differences in Hsp70 function required for growth and [PSI(+)] propagation and suggesting that Hsp70 interacts differently with [PSI(+)] prion aggregates than with other cellular substrates. Complementary suppression of altered activity between forward and suppressing mutations suggests that mutations that impair [PSI(+)] affect a similar Hsp70 function and that suppressing mutations similarly overcome this effect. All new mutations that impaired [PSI(+)] propagation were located in the ATPase domain. Locations and homology of several suppressing substitutions suggest that they weaken Hsp70's substrate-trapping conformation, implying that impairment of [PSI(+)] by forward mutations is due to altered ability of the ATPase domain to regulate substrate binding. Other suppressing mutations are in residues important for interactions with Hsp40 or TPR-containing cochaperones, suggesting that such interactions are necessary for the impairment of [PSI(+)] propagation caused by mutant Ssa1p.

Hsp90 enables Ctf13p/Skp1p to nucleate the budding yeast kinetochore.

Binding of CBF3, a protein complex consisting of Ndc10p, Cep3p, Ctf13p, and Skp1p, to the centromere DNA nucleates kinetochore formation in budding yeast. Here, we investigate how the Ctf13p/Skp1p complex becomes competent to form the CBF3-centromere DNA complex. As revealed by mass spectrometry, Ctf13p and Skp1p carry two and four phosphate groups, respectively. Complete dephosphorylation of Ctf13p and Skp1p does not interfere with the formation of CBF3-centromere DNA complexes in vitro. Furthermore, deletion of corresponding phosphorylation sites results in viable cells. Thus, in contrast to the current view, phosphorylation of Ctf13p and Skp1p is not essential for the formation of CBF3-centromere DNA complexes. Instead, the formation of active Ctf13p/Skp1p requires Hsp90. Several lines of evidence support this conclusion: activation of heterologous Ctf13p/Skp1p by reticulocyte lysate is inhibited by geldanamycin and Hsp90 depletion. skp1 mutants exhibit growth defects on media containing geldanamycin. A skp1 mutation together with Hsp90 mutations exhibits synthetic lethality. An Hsp90 mutant contains decreased levels of active Ctf13p/Skp1p.

The Molecular Chaperone Hsp90 Mediates Heme Activation of the Yeast Transcriptional Activator Hap1

Hsp90 plays critical roles in the proper functioning of a wide array of eukaryotic signal transducers such as steroid receptors and tyrosine kinases. Hap1 is a naturally occurring substrate of Hsp90 in Saccharomyces cerevisiae. Hap1 transcriptional activity is precisely and stringently controlled by heme. Previous biochemical studies suggest that in the absence of heme, Hap1 is bound to Hsp90 and other proteins, forming a higher order complex termed HMC (high molecular weight complex), and is repressed. Heme promotes the disruption of the HMC and activates Hap1, permitting Hap1 to bind to DNA with high affinity and to stimulate transcription. By lowering the expression levels of wild-type Hsp90, using a highly specific Hsp90 inhibitor, and by examining the effects of various Hsp90 mutants on Hap1, we show that Hsp90 is critical for Hap1 activation by heme. Furthermore, we show that many Hsp90 mutants exert differential effects on Hap1 and steroid receptors. Notably, mutant G313N weakens Hsp90 steroid receptor interaction but strongly enhances Hsp90-Hap1 interaction and increases Hap1 resistance to protease digestion. Additionally, we found that a heme-independent Hap1 mutant still depends on Hsp90 for high activity. These experiments together suggest that Hsp90 promotes Hap1 activation by inducing or maintaining Hap1 in a transcriptionally active conformation.

Regulation of Heat Shock Protein 90 ATPase Activity by Sequences in the Carboxyl Terminus

Hsp90, in addition to being an abundant and pivotal cytoplasmic chaperone protein, has been shown to be a weak ATPase. In an effort to characterize the ATPase activity of hsp90, we have observed marked differences in activities among various species of hsp90. Chicken or human hsp90 hydrolyzed ATP with a k(cat) of 0.02 min(-1) and a K(m) greater than 300 microm. In contrast, yeast hsp90 and TRAP1, an hsp90 homologue found in mitochondria, were 10-100-fold more active as ATPases. Sedimentation studies confirmed that all are dimeric proteins. Chicken hsp90 mutants were then analyzed to identify regions within the protein that influence ATPase activity. A truncation mutant of chicken hsp90, N1-573, was found to be monomeric, and yet the catalytic efficiency (k(cat)/K(m)) was greater than 100 times that of the full-length protein (k(cat) of 0.24 min(-1) and K(m) of 60 microm). In contrast, an internal deletion mutant, Delta661-677, was also monomeric but failed to hydrolyze ATP. Finally, deletion of the last 30 amino acids resulted in a dimeric protein with an ATPase activity very similar to full-length hsp90. These data indicate that sequences within the last one-fourth of hsp90 regulate ATP hydrolysis.

Loss of the mitochondrial Hsp70 functions causes aggregation of mitochondria in yeast cells.

Ssc1p, a member of the Hsp70 family in the mitochondrial matrix of budding yeast, mediates protein import into mitochondria and prevents irreversible aggregation of proteins in the mitochondrial matrix during folding/assembly or at elevated temperature. Here, we show that functional inactivation of the mitochondrial Hsp70 system causes aggregation of mitochondria. When temperature-sensitive mitochondrial Hsp70 mutant cells were incubated at restrictive temperature, a tubular network of mitochondria was collapsed to form aggregates. Inhibition of protein synthesis in the cytosol did not suppress the mitochondrial aggregation and functional impairment of Tim23, a subunit of mitochondrial protein translocator in the inner membrane, did not cause mitochondrial aggregation. Therefore defects of the Hsp70 function in protein import into mitochondria or resulting accumulation of precursor forms of mitochondrial proteins outside the mitochondria are not the causal reason for the aberrant mitochondrial morphology. By contrast, deletion of Mdj1p, a functional partner for mitochondrial Hsp70 in prevention of irreversible protein aggregation in the matrix, but not in protein import into mitochondria, caused aggregation of mitochondria, which was enhanced at elevated temperature (37 degrees C). The aggregation of mitochondria at 37 degrees C was reversed when the temperature was lowered to 23 degrees C unless protein synthesis was blocked. On the basis of these results, we propose that the mitochondrial matrix contains a protein that is responsible for the maintenance of mitochondrial morphology and requires mitochondrial Hsp70 for its function.

Hsp90 chaperone complexes are required for the activity and stability of yeast protein kinases Mik1, Wee1 and Swe1

The Wee1 protein kinase negatively regulates entry into mitosis by mediating the inhibitory tyrosine phosphorylation of Cdc2-cyclin B kinase. The stability and activity of Wee1 from the fission yeast Schizosaccharomyces pombe is critically dependent on functional Hsp90 chaperones. Here we identify two related tyrosine protein kinases, Mik1 from fission yeast and its Saccharomyces cerevisiae homolog Swe1, as Hsp90 substrates and show that the kinase domain is sufficient to mediate this interaction. Morphological and biochemical defects arising from overexpression of the kinases in fission yeast are suppressed in the conditional Hsp90 mutant swo1-26. A subset of all three kinases is associated with the Hsp90 cochaperones cyclophilin 40 and p23. Under conditions of impaired chaperone function or treatment with the Hsp90 inhibitory drug geldanamycin, intracellular levels of the kinases are reduced and the proteins become rapidly degraded by the proteasome machinery, indicating that Wee1, Mik1 and Swe1 require Hsp90 heterocomplexes for their stability and maintenance of function.

Polypeptide release by Hsp90 involves ATP hydrolysis and is enhanced by the co-chaperone p23.

The molecular chaperone Hsp90 binds and hydrolyses ATP, but how this ATPase activity regulates the interaction of Hsp90 with a polypeptide substrate is not yet understood. Using the glucocorticoid receptor ligand binding domain as a substrate, we show that dissociation of Hsp90 from bound polypeptide depends on the Hsp90 ATPase and is blocked by geldanamycin, a specific ATPase inhibitor. The co-chaperone p23 greatly stimulates Hsp90 substrate release with ATP, but not with the non-hydrolysable nucleotides ATPgammaS or AMP-PNP. Point mutants of Hsp90 with progressively lower ATPase rates are progressively slower in ATP-dependent substrate release but are still regulated by p23. In contrast, ATPase-inactive Hsp90 mutants release substrate poorly and show no p23 effect. These results outline an ATP-driven cycle of substrate binding and release for Hsp90 which differs from that of other ATP-driven chaperones. Conversion of the ATP state of Hsp90 to the ADP state through hydrolysis is required for efficient release of substrate polypeptide. p23 couples the ATPase activity to polypeptide dissociation and thus can function as a substrate release factor for Hsp90.