Structural and functional analysis of SGT1-HSP90 core complex required for innate immunity in plants

SGT1 (Suppressor of G2 allele of skp1), a co-chaperone of HSP90 (Heat-shock protein 90), is required for innate immunity in plants and animals. Unveiling the cross talks between SGT1 and other co-chaperones such as p23, AHA1 (Activator of HSP90 ATPase 1) or RAR1 (Required for Mla12 resistance) is an important step towards understanding the HSP90 machinery. Nuclear magnetic resonance spectroscopy and mutational analyses of HSP90 revealed the nature of its binding with the CS domain of SGT1. Although CS is structurally similar to p23, these domains were found to non-competitively bind to various regions of HSP90; yet, unexpectedly, full-length SGT1 could displace p23 from HSP90. RAR1 partly shares the same binding site with HSP90 as the CS domain, whereas AHA1 does not. This analysis allowed us to build a structural model of the HSP90–SGT1 complex and to obtain a compensatory mutant pair between both partners that is able to restore virus resistance in vivo through Rx (Resistance to potato virus X) immune sensor stabilization.     

SGT1 is not required for plant LRR-RLK-mediated immunity

Plant immune signalling activated by the perception of pathogen-associated molecular patterns (PAMPs) or effector proteins is mediated by pattern-recognition receptors (PRRs) and nucleotide-binding and leucine-rich repeat domain-containing receptors (NLRs), which often share cellular components and downstream responses. Many PRRs are leucine-rich repeat receptor-like kinases (LRR-RLKs), which mostly perceive proteinaceous PAMPs. The suppressor of the G2 allele of skp1 (SGT1) is a core immune regulator required for the activation of NLR-mediated immunity. In this work, we examined the requirement of SGT1 for immune responses mediated by several LRR-RLKs in both Nicotiana benthamiana and Arabidopsis. Using complementary genetic approaches, we found that SGT1 is not limiting for early PRR-dependent responses or antibacterial immunity. We therefore conclude that SGT1 does not play a significant role in bacterial PAMP-triggered immunity.     

Role of SGT1 in resistance protein accumulation in plant immunity

A highly conserved eukaryotic protein SGT1 binds specifically to the molecular chaperone, HSP90. In plants, SGT1 positively regulates disease resistance conferred by many Resistance (R) proteins and developmental responses to the phytohormone, auxin. We show that silencing of SGT1 in Nicotiana benthamiana causes a reduction in steady-state levels of the R protein, Rx. These data support a role of SGT1 in R protein accumulation, possibly at the level of complex assembly. In Arabidopsis, two SGT1 proteins, AtSGT1a and AtSGT1b, are functionally redundant early in development. AtSGT1a and AtSGT1b are induced in leaves upon infection and either protein can function in resistance once a certain level is attained, depending on the R protein tested. In unchallenged tissues, steady-state AtSGT1b levels are at least four times greater than AtSGT1a. While the respective tetratricopeptide repeat (TPR) domains of SGT1a and SGT1b control protein accumulation, they are dispensable for intrinsic functions of SGT1 in resistance and auxin responses.     

Expression of RPS4 in tobacco induces an AvrRps4-independent HR that requires EDS1, SGT1 and HSP90

The Arabidopsis RPS4 gene belongs to the Toll/interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR-NB-LRR) class of plant resistance (R) genes. It confers resistance to Pseudomonas syringae carrying the avirulence gene avrRps4. Transient expression of genomic RPS4 driven by the 35S promoter in tobacco leaves induces an AvrRps4-independent hypersensitive response (HR). The same phenotype is seen after expression of a full-length RPS4 cDNA. This indicates that alternative splicing of RPS4 is not involved in this HR. The extent of HR is correlated with RPS4 protein levels. Deletion analyses of RPS4 domains show the TIR domain is required for the HR phenotype. Mutations in the P-loop motif of the NB domain abolish the HR. Using virus-induced gene silencing, we found that the cell death resulting from RPS4 expression is dependent on the three plant signalling components EDS1, SGT1 and HSP90. All these data suggest that heterologous expression of an R gene can result in activation of cell death even in the absence of its cognate avirulence product, and provides a system for studying the RPS4 domains required for HR.     

Structural elements of ADP-ribosylation factor 1 required for functional interaction with cytohesin-1

ADP-ribosylation factor 1 (ARF1) is a 20-kDa guanine nucleotide-binding protein involved in vesicular trafficking. Conversion of inactive ARF-GDP to active ARF-GTP is catalyzed by guanine nucleotide exchange proteins such as cytohesin-1. Cytohesin-1 and its Sec7 domain (C-1Sec7) exhibit guanine nucleotide exchange protein activity with ARF1 but not ARF-like protein 1 (ARL1), which is 57% identical in amino acid sequence. With chimeric proteins composed of ARF1 (F) and ARL1 (L) sequences we identified three structural elements responsible for this specificity. Cytohesin-1 increased [35S]guanosine 5'-(gamma-thio)triphosphate binding to L28/F (first 28 residues of L, remainder F) and to a much lesser extent F139/L, and mut13F139/L (F139/L with random sequence in the first 13 positions) but not Delta13ARF1 that lacks the first 13 amino acids; therefore, a nonspecific ARF N terminus was required for cytohesin-1 action. The N terminus was not, however, required for that of C-1Sec7. Both C-1Sec7 and cytohesin-1 effectively released guanosine 5'-(gamma-thio)triphosphate from ARF1, but only C-1Sec7 displaced the nonhydrolyzable GTP analog bound to mut13F139/L, again indicating that structure in addition to the Sec7 domain is involved in cytohesin-1 interaction. Some element(s) of the C-terminal region is also involved, because replacement of the last 42 amino acids with ARL sequence in F139L decreased markedly the interaction with cytohesin-1. Participation of both termini is consistent with the crystallographic structure of ARF in which the two terminal alpha-helices are in close proximity. ARF1 residues 28-50 are also important in the interaction with cytohesin-1; replacement of Lys-38 with Gln, the corresponding residue in ARL1, abolished the ability to serve as substrate for cytohesin-1 or C-1Sec7. These studies have defined multiple structural elements in ARF1, including switch 1 and the N and C termini, that participate in functional interactions with cytohesin-1 (or its catalytic domain C-1Sec7), which were not apparent from crystallographic analysis.     

Structural and functional characterization of human SGT and its interaction with Vpu of the human immunodeficiency virus type 1

The small glutamine-rich tetratricopeptide repeat protein (SGT) belongs to a family of cochaperones that interacts with both Hsp70 and Hsp90 via the so-called TPR domain. Here, we present the crystal structure of the TPR domain of human SGT (SGT-TPR), which shows that it contains typical features found in the structures of other TPR domains. Previous studies show that full-length SGT can bind to both Vpu and Gag of human immunodeficiency virus type 1 (HIV-1) and the overexpression of SGT in cells reduces the efficiency of HIV-1 particle release. We show that SGT-TPR can bind Vpu and reduce the amount of HIV-1 p24, which is the viral capsid, secreted from cells transfected with the HIV-1 proviral construct, albeit at a lower efficiency than full-length SGT. This indicates that the TPR domain of SGT is sufficient for the inhibition of HIV-1 particle release but the N- and/or C-terminus also have some contributions. The SGT binding site in Vpu was also identified by using peptide array and confirmed by GST pull-down assay.     

A rice gid1 suppressor mutant reveals that gibberellin is not always required for interaction between its receptor, GID1, and DELLA proteins

To investigate gibberellin (GA) signaling using the rice (Oryza sativa) GA receptor GIBBERELLIN-INSENSITIVE DWARF1 (GID1) mutant gid1-8, we isolated a suppressor mutant, Suppressor of gid1-1 (Sgd-1). Sgd-1 is an intragenic mutant containing the original gid1-8 mutation (L45F) and an additional amino acid substitution (P99S) in the loop region. GID1(P99S) interacts with the rice DELLA protein SLENDER RICE1 (SLR1), even in the absence of GA. Substitution of the 99th Pro with other amino acids revealed that substitution with Ala (P99A) caused the highest level of GA-independent interaction. Physicochemical analysis using surface plasmon resonance revealed that GID1(P99A) has smaller K(a) (association) and K(d) (dissociation) values for GA(4) than does wild-type GID1. This suggests that the GID1(P99A) lid is at least partially closed, resulting in both GA-independent and GA-hypersensitive interactions with SLR1. One of the three Arabidopsis thaliana GID1s, At GID1b, can also interact with DELLA proteins in the absence of GA, so we investigated whether GA-independent interaction of At GID1b depends on a mechanism similar to that of rice GID1(P99A). Substitution of the loop region or a few amino acids of At GID1b with those of At GID1a diminished its GA-independent interaction with GAI while maintaining the GA-dependent interaction. Soybean (Glycine max) and Brassica napus also have GID1s similar to At GID1b, indicating that these unique GID1s occur in various dicots and may have important functions in these plants.     

HSP90 is required for TAK1 stability but not for its activation in the pro-inflammatory signaling pathway

The protein kinase transforming-growth-factor-β-activated kinase-1 (TAK1) is a key regulator in the pro-inflammatory signaling pathway and is activated by tumor necrosis factor-α, interleukin-1 (IL-1) and lipopolysaccharide (LPS). We describe the identification of TAK1 as a client protein of the 90 kDa heat-shock protein (Hsp90)/cell division cycle protein 37 (Cdc37) chaperones. However, Hsp90 is not required for the activation of TAK1 as short exposure to the Hsp90 inhibitor, 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) did not affect its activation by LPS or IL-1. Prolonged treatment of cells with 17-AAG inhibits Hsp90 and downregulates TAK1. Our results suggest that Hsp90 is required for the folding and stability of TAK1 but is displaced and no longer required when TAK1 is complexed to TAK1-binding protein-1 (TAB1).

Structured summary

MINT-6797182:

TAK1 (uniprotkb:O43318-2) physically interacts (MI:0218) with CDC37 (uniprotkb:Q16543) and HSP90 (uniprotkb:P07900) by anti bait coimmunoprecipitation (MI:0006)

MINT-6797194:

TAK1 (uniprotkb:O43318-2) physically interacts (MI:0218) with TAB1 (uniprotkb:Q15750), HSP90 (uniprotkb:P07900) and CDC37 (uniprotkb:Q16543) by anti bait coimmunoprecipitation (MI:0006)

MINT-6797248:

TAK1 (uniprotkb:Q62073) physically interacts (MI:0218) with HSP90 (uniprotkb:P07901), CDC37 (uniprotkb:Q61081), TAB2 (uniprotkb:Q99K90) and TAB1 (uniprotkb:Q8CF89) by anti bait coimmunoprecipitation (MI:0006)

MINT-6797232:

TAK1 (uniprotkb:O43318-2) physically interacts (MI:0218) with HSP90 (uniprotkb:P07900) and CDC37 (uniprotkb:Q16543) by pull down (MI:0096)

MINT-6797216:

TAK1 (uniprotkb:O43318-2) physically interacts (MI:0218) with TAB2 (uniprotkb:Q9NYJ8), CDC37 (uniprotkb:Q16543), HSP90 (uniprotkb:P07900) and TAB1 (uniprotkb:Q15750) by anti bait coimmunoprecipitation (MI:0006)

     

Interaction of heat-shock protein 90 beta isoform (HSP90 beta) with cellular inhibitor of apoptosis 1 (c-IAP1) is required for cell differentiation

Members of the inhibitor of apoptosis protein (IAP) family have demonstrated functions in cell death, cell signalling, cell migration and mitosis. Several of them are E3 enzymes in the ubiquitination of proteins that leads to their degradation by the proteosomal machinery. We previously reported that one of them, cellular inhibitor of apoptosis protein-1 (c-IAP1), migrated from the nucleus to the surface of the Golgi apparatus in cells undergoing differentiation. Here, we show that c-IAP1 is a client protein of the stress protein HSP90 beta. In three distinct cellular models, the two proteins interact and migrate from the nucleus to the cytoplasm along the differentiation process through a leptomycin B-sensitive pathway. Inhibition of HSP90 proteins by small chemical molecules and specific depletion of HSP90 beta isoform by siRNA both lead to auto-ubiquitination of c-IAP1 and its degradation by the proteasome machinery. This chaperone function of HSP90 towards c-IAP1 is specific of its beta isoform as specific depletion of HSP90alpha does not affect c-IAP1 content. Chemical inhibition of HSP90 or siRNA-mediated depletion of HSP90 beta both inhibit cell differentiation, which can be reproduced by siRNA-mediated depletion of c-IAP1. Altogether, these results suggest that HSP90 beta prevents auto-ubiquitination and degradation of its client protein c-IAP1, whose depletion would be sufficient to inhibit cell differentiation.     

The interaction between Sgt1p and Skp1p is regulated by HSP90 chaperones and is required for proper CBF3 assembly

Sgt1p is a well-conserved protein proposed to be involved in a number of cellular processes. Genetic studies of budding yeast suggest a role for SGT1 in signal transduction, cell cycle advance, and chromosome segregation. Recent evidence has linked Sgt1p to HSP90 chaperones, although the precise relationship between these proteins is unclear. To further explore the role of Sgt1p in these processes, we have characterized the interactions among Sgt1p, the inner kinetochore complex CBF3, and HSP90 chaperones. We show that the amino terminus of Sgt1p interacts with CBF3 subunits Skp1p and Ctf13p. HSP90 interacts with Sgt1p and, in combination with the carboxy terminus of Sgt1p, regulates the interaction between Sgt1p and Skp1p in a nucleotide-dependent manner. While the Sgt1p-Skp1p interaction is required for CBF3 assembly, mutations that stabilize this interaction prevent the turnover of protein complexes important for CBF3 assembly. We propose that HSP90 and Sgt1p act together as a molecular switch, maintaining transient interactions required to balance protein complex assembly with turnover.     

Structural and functional analysis of a plant resistance protein TIR domain reveals interfaces for self-association, signaling, and autoregulation

The Toll/interleukin-1 receptor (TIR) domain occurs in animal and plant immune receptors. In the animal Toll-like receptors, homodimerization of the intracellular TIR domain is required for initiation of signaling cascades leading to innate immunity. By contrast, the role of the TIR domain in cytoplasmic nucleotide-binding/leucine-rich repeat (NB-LRR) plant immune resistance proteins is poorly understood. L6 is a TIR-NB-LRR resistance protein from flax (Linum usitatissimum) that confers resistance to the flax rust phytopathogenic fungus (Melampsora lini). We determine the crystal structure of the L6 TIR domain and show that, although dispensable for pathogenic effector protein recognition, the TIR domain alone is both necessary and sufficient for L6 immune signaling. We demonstrate that the L6 TIR domain self-associates, most likely forming?a homodimer. Analysis of the structure combined with site-directed mutagenesis suggests that self-association is a requirement for immune signaling and reveals distinct surface regions involved in self-association, signaling, and autoregulation.     

Nucleocytoplasmic distribution is required for activation of resistance by the potato NB-LRR receptor Rx1 and is balanced by its functional domains

The Rx1 protein, as many resistance proteins of the nucleotide binding-leucine-rich repeat (NB-LRR) class, is predicted to be cytoplasmic because it lacks discernable nuclear targeting signals. Here, we demonstrate that Rx1, which confers extreme resistance to Potato virus X, is located both in the nucleus and cytoplasm. Manipulating the nucleocytoplasmic distribution of Rx1 or its elicitor revealed that Rx1 is activated in the cytoplasm and cannot be activated in the nucleus. The coiled coil (CC) domain was found to be required for accumulation of Rx1 in the nucleus, whereas the LRR domain promoted the localization in the cytoplasm. Analyses of structural subdomains of the CC domain revealed no autonomous signals responsible for active nuclear import. Fluorescence recovery after photobleaching and nuclear fractionation indicated that the CC domain binds transiently to large complexes in the nucleus. Disruption of the Rx1 resistance function and protein conformation by mutating the ATP binding phosphate binding loop in the NB domain, or by silencing the cochaperone SGT1, impaired the accumulation of Rx1 protein in the nucleus, while Rx1 versions lacking the LRR domain were not affected in this respect. Our results support a model in which interdomain interactions and folding states determine the nucleocytoplasmic distribution of Rx1.     

Farnesylation of Ydj1 is required for in vivo interaction with Hsp90 client proteins

Ydj1 of Saccharomyces cerevisiae is an abundant cytosolic Hsp40, or J-type, molecular chaperone. Ydj1 cooperates with Hsp70 of the Ssa family in the translocation of preproteins to the ER and mitochondria and in the maturation of Hsp90 client proteins. The substrate-binding domain of Ydj1 directly interacts with steroid receptors and is required for the activity of diverse Hsp90-dependent client proteins. However, the effect of Ydj1 alteration on client interaction was unknown. We analyzed the in vivo interaction of Ydj1 with the protein kinase Ste11 and the glucocorticoid receptor. Amino acid alterations in the proposed client-binding domain or zinc-binding domain had minor effects on the physical interaction of Ydj1 with both clients. However, alteration of the carboxy-terminal farnesylation signal disrupted the functional and physical interaction of Ydj1 and Hsp90 with both clients. Similar effects were observed upon deletion of RAM1, which encodes one of the subunits of yeast farnesyltransferase. Our results indicate that farnesylation is a major factor contributing to the specific requirement for Ydj1 in promoting proper regulation and activation of diverse Hsp90 clients.     

Molecular characterization of functional domains in the protein kinase SOS2 that is required for plant salt tolerance

The SOS3 (for SALT OVERLY SENSITIVE3) calcium binding protein and SOS2 protein kinase are required for sodium and potassium ion homeostasis and salt tolerance in Arabidopsis. We have shown previously that SOS3 interacts with and activates the SOS2 protein kinase. We report here the identification of a SOS3 binding motif in SOS2 that also serves as the kinase autoinhibitory domain. Yeast two-hybrid assays as well as in vitro binding assays revealed a 21-amino acid motif in the regulatory domain of SOS2 that is necessary and sufficient for interaction with SOS3. Database searches revealed a large family of SOS2-like protein kinases containing such a SOS3 binding motif. Using a yeast two-hybrid system, we show that these SOS2-like kinases interact with members of the SOS3 family of calcium binding proteins. Two-hybrid assays also revealed interaction between the N-terminal kinase domain and the C-terminal regulatory domain within SOS2, suggesting that the regulatory domain may inhibit kinase activity by blocking substrate access to the catalytic site. Removal of the regulatory domain of SOS2, including the SOS3 binding motif, resulted in constitutive activation of the protein kinase, indicating that the SOS3 binding motif can serve as a kinase autoinhibitory domain. Constitutively active SOS2 that is SOS3 independent also was produced by changing Thr(168) to Asp in the activation loop of the SOS2 kinase domain. Combining the Thr(168)-to-Asp mutation with the autoinhibitory domain deletion created a superactive SOS2 kinase. These results provide insights into regulation of the kinase activities of SOS2 and the SOS2 family of protein kinases.     

A RanGAP protein physically interacts with the NB-LRR protein Rx, and is required for Rx-mediated viral resistance

Race-specific disease resistance in plants is mediated by the products of host disease resistance (R) genes. Plant genomes possess hundreds of R gene homologs encoding nucleotide-binding and leucine-rich repeat (NB-LRR) proteins. NB-LRR proteins induce a disease resistance response following recognition of pathogen-encoded avirulence (Avr) proteins. However, little is known about the general mechanisms by which NB-LRR proteins recognize Avr proteins or how they subsequently induce defense responses. The Rx NB-LRR protein of potato confers resistance to potato virus X (PVX). Using a co-purification strategy, we have identified a Ran GTPase-activating protein (RanGAP2) as an Rx-interacting protein. We show by co-immunoprecipitation that this interaction is mediated in planta through the putative signaling domain at the Rx amino terminus. Overexpression of RanGAP2 results in activation of certain Rx derivatives. Likewise, knocking down RanGAP2 expression in Nicotiana benthamiana by virus-induced gene silencing compromises Rx-mediated resistance to PVX. Thus, we have demonstrated a novel role for a RanGAP in the function of a plant disease resistance response.     

Structural and functional analysis show that the Escherichia coli uncharacterized protein YjcS is likely an alkylsulfatase

Sodium dodecyl sulfate (SDS) is a widely used anionic surfactant in industry and research settings, and is known to have a detrimental effect to the environment. The pathway of SDS degradation by bacteria is initiated by an alkylsulfatase and the oxidized product, 1-dodecanoic acid, subsequently enters into the β-oxidation pathway and is used as a carbon source. In this work, we solved the crystal structure of Escherichia coli uncharacterized protein YjcS and identified that it belongs to the Type III alkylsulfatase with a signal peptide (residues 1–29) at the N terminus. YjcS hydrolyzed SDS and the double mutant D184N-H185A located in the conserved HXHXDH catalytic motif abolished this activity.     

Perturbation with intrabodies reveals that calpain cleavage is required for degradation of huntingtin exon 1

Background

Proteolytic processing of mutant huntingtin (mHtt), the protein that causes Huntington''s disease (HD), is critical for mHtt toxicity and disease progression. mHtt contains several caspase and calpain cleavage sites that generate N-terminal fragments that are more toxic than full-length mHtt. Further processing is then required for the degradation of these fragments, which in turn, reduces toxicity. This unknown, secondary degradative process represents a promising therapeutic target for HD.

Methodology/Principal Findings

We have used intrabodies, intracellularly expressed antibody fragments, to gain insight into the mechanism of mutant huntingtin exon 1 (mHDx-1) clearance. Happ1, an intrabody recognizing the proline-rich region of mHDx-1, reduces the level of soluble mHDx-1 by increasing clearance. While proteasome and macroautophagy inhibitors reduce turnover of mHDx-1, Happ1 is still able to reduce mHDx-1 under these conditions, indicating Happ1-accelerated mHDx-1 clearance does not rely on these processes. In contrast, a calpain inhibitor or an inhibitor of lysosomal pH block Happ1-mediated acceleration of mHDx-1 clearance. These results suggest that mHDx-1 is cleaved by calpain, likely followed by lysosomal degradation and this process regulates the turnover rate of mHDx-1. Sequence analysis identifies amino acid (AA) 15 as a potential calpain cleavage site. Calpain cleavage of recombinant mHDx-1 in vitro yields fragments of sizes corresponding to this prediction. Moreover, when the site is blocked by binding of another intrabody, V_L12.3, turnover of soluble mHDx-1 in living cells is blocked.

Conclusions/Significance

These results indicate that calpain-mediated removal of the 15 N-terminal AAs is required for the degradation of mHDx-1, a finding that may have therapeutic implications.     

Structure of minimal tetratricopeptide repeat domain protein Tah1 reveals mechanism of its interaction with Pih1 and Hsp90

Tah1 and Pih1 are novel Hsp90 interactors. Tah1 acts as a cofactor of Hsp90 to stabilize Pih1. In yeast, Hsp90, Tah1, and Pih1 were found to form a complex that is required for ribosomal RNA processing through their effect on box C/D small nucleolar ribonucleoprotein assembly. Tah1 is a minimal tetratricopeptide repeat protein of 111 amino acid residues that binds to the C terminus of the Hsp90 molecular chaperone, whereas Pih1 consists of 344 residues of unknown fold. The NMR structure of Tah1 has been solved, and this structure shows the presence of two tetratricopeptide repeat motifs followed by a C helix and an unstructured region. The binding of Tah1 to Hsp90 is mediated by the EEVD C-terminal residues of Hsp90, which bind to a positively charged channel formed by Tah1. Five highly conserved residues, which form a two-carboxylate clamp that tightly interacts with the ultimate Asp-0 residue of the bound peptide, are also present in Tah1. Tah1 was found to bind to the C terminus of Pih1 through the C helix and the unstructured region. The C terminus of Pih1 destabilizes the protein in vitro and in vivo, whereas the binding of Tah1 to Pih1 allows for the formation of a stable complex. Based on our data, a model for an Hsp90-Tah1-Pih1 ternary complex is proposed.     

The carboxy-terminal region of mammalian HSP90 is required for its dimerization and function in vivo.

The majority of mouse HSP90 exists as alpha-alpha and beta-beta homodimers. Truncation of the 15-kDa carboxy-terminal region of mouse HSP90 by digestion with the Ca(2+)-dependent protease m-calpain caused dissociation of the dimer. When expressed in a reticulocyte lysate, the full-length human HSP90 alpha formed a dimeric form. A plasmid harboring human HSP90 alpha cDNA was constructed so that the carboxy-terminal 49 amino acid residues were removed when translated in vitro. This carboxy-terminally truncated human HSP90 alpha was found to exist as a monomer. In contrast, loss of the 118 amino acid residues from the amino terminus of human HSP90 alpha did not affect its in vitro dimerization. Introduction of an expression plasmid harboring the full-length human HSP90 alpha complements the lethality caused by the double mutations of two HSP90-related genes, hsp82 and hsc82, in a haploid strain of Saccharomyces cerevisiae. The carboxy-terminally truncated human HSP90 alpha neither formed dimers in yeast cells nor rescued the lethal double mutant.