Plastid HSP90C C-terminal extension region plays a regulatory role in chaperone activity and client binding.

HSP90Cs are essential molecular chaperones localized in the plastid stroma that maintain protein homeostasis and assist the import and thylakoid transport of chloroplast proteins. While HSP90C contains all conserved domains as an HSP90 family protein, it also possesses a unique feature in its variable C-terminal extension (CTE) region. This study elucidated the specific function of this HSP90C CTE region. Our phylogenetic analyses revealed that this intrinsically disordered region contains a highly conserved DPW motif in the green lineages. With biochemical assays, we showed that the CTE is required for the chaperone to effectively interact with client proteins PsbO1 and LHCB2 to regulate ATP-independent chaperone activity and to effectuate its ATP hydrolysis. The CTE truncation mutants could support plant growth and development reminiscing the wild type under normal conditions except for a minor phenotype in cotyledon when expressed at a level comparable to wild type. However, higher HSP90C expression was observed to correlate with a stronger response to specific photosystem II inhibitor DCMU, and CTE truncations dampened the response. Additionally, when treated with lincomycin to inhibit chloroplast protein translation, CTE truncation mutants showed a delayed response to PsbO1 expression repression, suggesting its role in chloroplast retrograde signaling. Our study therefore provides insights into the mechanism of HSP90C in client protein binding and the regulation of green chloroplast maturation and function, especially under stress conditions.

Plastid Molecular Chaperone HSP90C Interacts with the SecA1 Subunit of Sec Translocase for Thylakoid Protein Transport.

The plastid stroma-localized chaperone HSP90C plays a crucial role in maintaining optimal proteostasis within chloroplasts and participates in protein translocation processes. While existing studies have revealed HSP90C's direct interaction with the Sec translocase-dependent client pre-protein PsbO1 and the SecY1 subunit of the thylakoid membrane-bound Sec1 translocase channel system, its direct involvement with the extrinsic homodimeric Sec translocase subunit, SecA1, remains elusive. Employing bimolecular fluorescence complementation (BiFC) assay and other in vitro analyses, we unraveled potential interactions between HSP90C and SecA1. Our investigation revealed dynamic interactions between HSP90C and SecA1 at the thylakoid membrane and stroma. The thylakoid membrane localization of this interaction was contingent upon active HSP90C ATPase activity, whereas their stromal interaction was associated with active SecA1 ATPase activity. Furthermore, we observed a direct interaction between these two proteins by analyzing their ATP hydrolysis activities, and their interaction likely impacts their respective functional cycles. Additionally, using PsbO1, a model Sec translocase client pre-protein, we studied the intricacies of HSP90C's possible involvement in pre-protein translocation via the Sec1 system in chloroplasts. The results suggest a complex nature of the HSP90C-SecA1 interaction, possibly mediated by the Sec client protein. Our studies shed light on the nuanced aspects of HSP90C's engagement in orchestrating pre-protein translocation, and we propose a potential collaborative role of HSP90C with SecA1 in actively facilitating pre-protein transport across the thylakoid membrane.

Knockout of endoplasmic reticulum-localized molecular chaperone HSP90.7 impairs seedling development and cellular auxin homeostasis in Arabidopsis.

The Arabidopsis endoplasmic reticulum-localized heat shock protein HSP90.7 modulates tissue differentiation and stress responses; however, complete knockout lines have not been previously reported. In this study, we identified and analyzed a mutant allele, hsp90.7-1, which was unable to accumulate the HSP90.7 full-length protein and showed seedling lethality. Microscopic analyses revealed its essential role in male and female fertility, trichomes and root hair development, proper chloroplast function, and apical meristem maintenance and differentiation. Comparative transcriptome and proteome analyses also revealed the role of the protein in a multitude of cellular processes. Particularly, the auxin-responsive pathway was specifically downregulated in the hsp90.7-1 mutant seedlings. We measured a much-reduced auxin content in both root and shoot tissues. Through comprehensive histological and molecular analyses, we confirmed PIN1 and PIN5 accumulations were dependent on the HSP90 function, and the TAA-YUCCA primary auxin biosynthesis pathway was also downregulated in the mutant seedlings. This study therefore not only fulfilled a gap in understanding the essential role of HSP90 paralogs in eukaryotes but also provided a mechanistic insight on the ER-localized chaperone in regulating plant growth and development via modulating cellular auxin homeostasis.

Cooperative Effect of ZIF-67-Derived Hollow NiCo-LDH and MoS2 on Enhancing the Flame Retardancy of Thermoplastic Polyurethane.

In this work, a novel three-dimensional (3D) hollow nickel-cobalt layered double hydroxide (NiCo-LDH) was synthesized using zeolitic imidazole framework-67 (ZIF-67) as a template, and then utilized to functionalize molybdenum disulfide (NiCo-LDH/MoS2) via electrostatic force. Flame retardant thermoplastic polyurethane (TPU) composites were prepared by the melt blending method. Compared to pure TPU, NiCo-LDH/MoS2 filled TPU composite was endowed with a decrease of 30.9% and 55.7% of the peak heat release rate (PHRR) and the peak smoke production rate (PSPR), respectively. Furthermore, the addition of NiCo-LDH/MoS2 can significantly improve the thermal stability and char yield of the TPU composite. The catalytic carbonization effect and dilution effect of NiCo-LDH, and the barrier effect of MoS2 nanosheets enable TPU composites with excellent flame retardancy and toxic gas suppression ability.

Oxidative and salt stresses alter the 26S proteasome holoenzyme and associated protein profiles in Arabidopsis thaliana.

BACKGROUND: The 26S proteasome, canonically composed of multi-subunit 19S regulatory (RP) and 20S core (CP) particles, is crucial for cellular proteostasis. Proteasomes are re-modeled, activated, or re-localized and this regulation is critical for plants in response to environmental stresses. The proteasome holoenzyme assembly and dissociation are therefore highly dynamic in vivo. However, the stoichiometric changes of the plant proteasomes and how proteasome associated chaperones vary under common abiotic stresses have not been systematically studied. RESULTS: Here, we studied the impact of abiotic stresses on proteasome structure, activity, and interacting partners in Arabidopsis thaliana. We analyzed available RNA expression data and observed that expressions of proteasome coding genes varied substantially under stresses; however, the protein levels of a few key subunits did not change significantly within 24 h. Instead, a switch in the predominant proteasome complex, from 26S to 20S, occurs under oxidative or salt stress. Oxidative stress also reduced the cellular ATP content and the association of HSP70-family proteins to the 20S proteasome, but enhanced the activity of cellular free form CP. Salt stress, on the other hand, did not affect cellular ATP level, but caused subtle changes in proteasome subunit composition and impacted bindings of assembly chaperones. Analyses of an array of T-DNA insertional mutant lines highlighted important roles for several putative assembly chaperones in seedling establishment and stress sensitivity. We also observed that knockout of PBAC1, one of the α-ring assembly chaperones, resulted in reduced germination and tearing of the seed coat following sterilization. CONCLUSIONS: Our study revealed an evolutionarily conserved mechanism of proteasome regulation during oxidative stress, involving dynamic regulation of the holoenzyme formation and associated regulatory proteins, and we also identified a novel role of the PBAC1 proteasome assembly chaperone in seed coat development.

Plastid chaperone HSP90C guides precursor proteins to the SEC translocase for thylakoid transport.

Chloroplast stromal factors involved in regulating thylakoid protein targeting are poorly understood. We previously reported that in Arabidopsis thaliana, the stromal-localized chaperone HSP90C (plastid heat shock protein 90) interacted with the nuclear-encoded thylakoid lumen protein PsbO1 (PSII subunit O isoform 1) and suggested a role for HSP90C in aiding PsbO1 thylakoid targeting. Using in organello transport assays, particularly with model substrates naturally expressed in stroma, we showed that light, exogenous ATP, and HSP90C activity were required for Sec-dependent transport of green fluorescent protein (GFP) led by the PsbO1 thylakoid targeting sequence. Using a previously identified PsbO1T200A mutant, we provided evidence that a stronger interaction between HSP90C and PsbO1 better facilitated its stroma-thylakoid trafficking. We also demonstrated that SecY1, the channel protein of the thylakoid SEC translocase, specifically interacted with HSP90C in vivo. Inhibition of the chaperone ATPase activity suppressed the association of the PsbO1GFP-HSP90C complex with SecY1. Together with analyzing the expression and accumulation of a few other thylakoid proteins that utilize the SRP, TAT, or SEC translocation pathways, we propose a model in which HSP90C forms a guiding complex that interacts with thylakoid protein precursors and assists in their specific targeting to the thylakoid SEC translocon.

HSP90C interacts with PsbO1 and facilitates its thylakoid distribution from chloroplast stroma in Arabidopsis.

Arabidopsis plastidic HSP90C is an HSP90 family molecular chaperone that is required for chloroplast development and function. To understand the mechanism of action of HSP90C within the chloroplast, we conducted a yeast two-hybrid screening and revealed it interacts directly with the photosystem II extrinsic protein PsbO1, which performs a canonical function in the thylakoid lumen. To understand the biological significance of HSP90C-PsbO1 interaction, we investigated the role of HSP90C in modulating the stromal and thylakoid distribution of PsbO1GFP fusion protein. Fusion to GFP significantly delays the PsbO1 thylakoid transport and induces a variegation phenotype. Overexpression of HSP90C promotes the thylakoid distribution of PsbO1GFP and alleviates the leaf variegation. By tracking the chloroplast maturation during photomorphogenesis, we observed PsbO1GFP tends to form distinct fluorescent clusters within the stroma with delayed thylakoid membrane biogenesis, while HSP90C overexpression corrects these adverse effects. We also demonstrated that active HSP90C function is specifically required for stable accumulation of mature PsbO1GFP in thylakoid by using specific inhibitor geldanamycin. This study therefore not only identified novel HSP90C interactors, but also reports for the first time that PsbO1 enroute from the cytoplasm to thylakoid lumen is tightly regulated by the HSP90C chaperone complex in plastid stroma; whereas the proper HSP90C homeostasis is also critical for chloroplast maturation and function.

ABA-dependent inhibition of the ubiquitin proteasome system during germination at high temperature in Arabidopsis.

During germination, endogenous and environmental factors trigger changes in the transcriptome, translatome and proteome to break dormancy. In Arabidopsis thaliana, the ubiquitin proteasome system (UPS) degrades proteins that promote dormancy to allow germination. While research on the UPS has focused on the identification of proteasomal substrates, little information is known about the regulation of its activity. Here we characterized the activity of the UPS during dormancy release and maintenance by monitoring protein ubiquitination and degradation of two proteasomal substrates: Suc-LLVY-AMC, a well characterized synthetic substrate, and FUSCA3 (FUS3), a dormancy-promoting transcription factor degraded by the 26S proteasome. Our data indicate that proteasome activity and protein ubiquitination increase during imbibition at optimal temperature (21°C), and are required for seed germination. However, abscisic acid (ABA) and supraoptimal temperature (32°C) inhibit germination by dampening both protein ubiquitination and proteasome activity. Inhibition of UPS function by high temperature is reduced by the ABA biosynthesis inhibitor, fluridone, and in ABA biosynthetic mutants, suggesting that it is ABA dependent. Accordingly, inhibition of FUS3 degradation at 32°C is also dependent on ABA. Native gels show that inhibition of proteasome activity is caused by interference with the 26S/30S ratio as well as free 19S and 20S levels, impacting the proteasome degradation cycle. Transfer experiments show that ABA-mediated inhibition of proteasome activity at 21°C is restricted to the first 2 days of germination, a time window corresponding to seed sensitivity to environmental and ABA-mediated growth inhibition. Our data show that ABA and high temperature inhibit germination under unfavourable growth conditions by repressing the UPS.

The Proteasome Subunit Rpn8 Interacts with the Small Nucleolar RNA Protein (snoRNP) Assembly Protein Pih1 and Mediates Its Ubiquitin-independent Degradation in Saccharomyces cerevisiae.

Pih1 is a scaffold protein of the Rvb1-Rvb2-Tah1-Pih1 (R2TP) protein complex, which is conserved in fungi and animals. The chaperone-like activity of the R2TP complex has been implicated in the assembly of multiple protein complexes, such as the small nucleolar RNA protein complex. However, the mechanism of the R2TP complex activity in vivo and the assembly of the complex itself are still largely unknown. Pih1 is an unstable protein and tends to aggregate when expressed alone. The C-terminal fragment of Pih1 contains multiple destabilization factors and acts as a degron when fused to other proteins. In this study, we investigated Pih1 interactors and identified a specific interaction between Pih1 and the proteasome subunit Rpn8 in yeast Saccharomyces cerevisiae when HSP90 co-chaperone Tah1 is depleted. By analyzing truncation mutants, we identified that the C-terminal 30 amino acids of Rpn8 are sufficient for the binding to Pih1 C terminus. With in vitro and in vivo degradation assays, we showed that the Pih1 C-terminal fragment Pih1(282-344) is able to induce a ubiquitin-independent degradation of GFP. Additionally, we demonstrated that truncation of the Rpn8 C-terminal disordered region does not affect proteasome assembly but specifically inhibits the degradation of the GFP-Pih1(282-344) fusion protein in vivo and Pih1 in vitro We propose that Pih1 is a ubiquitin-independent proteasome substrate, and the direct interaction with Rpn8 C terminus mediates its proteasomal degradation.

A tetratricopeptide repeat domain-containing protein SSR1 located in mitochondria is involved in root development and auxin polar transport in Arabidopsis.

Auxin polar transport mediated by a group of Pin-formed (PIN) transporters plays important roles in plant root development. However, the mechanism underlying the PIN expression and targeting in response to different developmental and environmental stimuli is still not fully understood. Here, we report a previously uncharacterized gene SSR1, which encodes a mitochondrial protein with tetratricopeptide repeat (TPR) domains, and show its function in root development in Arabidopsis thaliana. In ssr1-2, a SSR1 knock-out mutant, the primary root growth was dramatically inhibited due to severely impaired cell proliferation and cell elongation. Significantly lowered level of auxin was found in ssr1-2 roots by auxin measurement and was further supported by reduced expression of DR5-driven reporter gene. As a result, the maintenance of the root stem cell niche is compromised in ssr1-2. It is further revealed that the expression level of several PIN proteins, namely, PIN1, PIN2, PIN3, PIN4 and PIN7, were markedly reduced in ssr1-2 roots. In particular, we showed that the reduced protein level of PIN2 on cell membrane in ssr1-2 is due to impaired retrograde trafficking, possibly resulting from a defect in retromer sorting system, which destines PIN2 for degradation in vacuoles. In conclusion, our results indicated that SSR1 is functioning in root development in Arabidopsis, possibly by affecting PIN protein expression and subcellular targeting.

A highly charged region in the middle domain of plant endoplasmic reticulum (ER)-localized heat-shock protein 90 is required for resistance to tunicamycin or high calcium-induced ER stresses.

Heat-shock protein 90 (HSP90) is a highly conserved molecular chaperone that is involved in modulating a multitude of cellular processes under both physiological and stress conditions. In Arabidopsis, there are seven HSP90 isoforms (HSP90.1-HSP90.7) that are localized in the cytoplasm/nucleus, mitochondrion, chloroplast, and endoplasmic reticulum (ER) where protein folding actively takes place. In this study, we analysed the sequence of ER-localized Arabidopsis HSP90.7 and the other ER GRP94 proteins from plants and animals, and identified a short, charged region that is specifically present in the middle domain of plant-derived GRP94 proteins. To understand the role of this charged region, we analysed transgenic plants that expressed a mutant protein, HSP90.7(Δ22), which had this charged region deleted. We showed that seedlings expressing HSP90.7(Δ22) had significantly enhanced sensitivity to ER stress induced by tunicamycin or a high concentration of calcium, although its general chaperone activity in preventing the model protein from heat-induced aggregation was not significantly affected. We also analysed the ATP-binding and hydrolysis activity of both wild-type and mutant HSP90.7 proteins, and found that they had slightly different ATP-binding affinities. Finally, using a yeast two-hybrid screen, we identified a small set of HSP90.7 interactors and showed that the charged region is not required for the candidate client interaction, although it may affect their binding affinity, thus providing potential targets for further investigation of HSP90.7 functions.

Cosuppression of the chloroplast localized molecular chaperone HSP90.5 impairs plant development and chloroplast biogenesis in Arabidopsis.

BACKGROUND: HSP90.5 is a chloroplast localized HSP90 family molecular chaperone in Arabidopsis, and it has been implicated in plant abiotic stress resistance, photomorphogenesis and nuclear-encoded protein import into the chloroplast. However, how these processes are controlled by HSP90 is not well understood. To understand the role of HSP90.5 in chloroplast function and biogenesis, in this study, we generated transgenic Arabidopsis plants that overexpress a C-terminally FLAG-tagged HSP90.5. By characterizing three HSP90.5 cosuppression lines, we demonstrated the essential role of HSP90.5 in plant growth and chloroplast biogenesis. RESULTS: Immunoblotting and quantitative PCR analyses revealed three independent HSP90.5 cosuppressing transgenic lines. All three cosuppression lines displayed a certain degree of variegated phenotype in photosynthetic tissues, and the cosuppression did not affect the expression of cytosolic HSP90 isoforms. HSP90.5 cosuppression was shown to be developmentally regulated and occurred mostly at late developmental stage in adult leaves and inflorescence tissues. HSP90.5 cosuppression also caused significantly reduced rosette leaf growth, transient starch storage, but did not affect rosette leaf initiation or inflorescence production, although the fertility was reduced. Isolation of chloroplasts and size exclusion chromatography analysis indicated that the FLAG at the HSP90.5 C-terminus does not affect its proper chloroplast localization and dimerization. Finally, transmission electron microscopy indicated that chloroplast development in HSP90.5 cosuppression leaves was significantly impaired and the integrity of chloroplast is highly correlated to the expression level of HSP90.5. CONCLUSION: We thoroughly characterized three HSP90.5 cosuppression lines, and demonstrated that properly controlled expression of HSP90.5 is required for plant growth and development in many tissues, and especially essential for chloroplast thylakoid formation. Since the homozygote of HSP90.5 knockout mutant is embryonically lethal, this study provides transgenic lines that mimic the conditional knockout line or siRNA line of the essential HSP90.5 gene in Arabidopsis.

The stability of the small nucleolar ribonucleoprotein (snoRNP) assembly protein Pih1 in Saccharomyces cerevisiae is modulated by its C terminus.

Pih1 is an unstable protein and a subunit of the R2TP complex that, in yeast Saccharomyces cerevisiae, also contains the helicases Rvb1, Rvb2, and the Hsp90 cofactor Tah1. Pih1 and the R2TP complex are required for the box C/D small nucleolar ribonucleoprotein (snoRNP) assembly and ribosomal RNA processing. Purified Pih1 tends to aggregate in vitro. Molecular chaperone Hsp90 and its cochaperone Tah1 are required for the stability of Pih1 in vivo. We had shown earlier that the C terminus of Pih1 destabilizes the protein and that the C terminus of Tah1 binds to the Pih1 C terminus to form a stable complex. Here, we analyzed the secondary structure of the Pih1 C terminus and identified two intrinsically disordered regions and five hydrophobic clusters. Site-directed mutagenesis indicated that one predicted intrinsically disordered region IDR2 is involved in Tah1 binding, and that the C terminus of Pih1 contains multiple destabilization or degron elements. Additionally, the Pih1 N-terminal domain, Pih1(1-230), was found to be able to complement the physiological role of full-length Pih1 at 37 °C. Pih1(1-230) as well as a shorter Pih1 N-terminal fragment Pih1(1-195) is able to bind Rvb1/Rvb2 heterocomplex. However, the sequence between the two disordered regions in Pih1 significantly enhances the Pih1 N-terminal domain binding to Rvb1/Rvb2. Based on these data, a model of protein-protein interactions within the R2TP complex is proposed.

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.

Expression of five AtHsp90 genes in Saccharomyces cerevisiae reveals functional differences of AtHsp90s under abiotic stresses.

The genome of Arabidopsis thaliana contains seven Hsp90 family genes. Three organellar and two cytosolic AtHsp90 isoforms were characterized by functionally expressing them in a temperature-sensitive Hsp90 mutant and a conditional Hsp90-null mutant of Saccharomyces cerevisiae. The cytosolic AtHsp90-1 and AtHsp90-2 showed function similar to that of yeast in chaperoning roles; they could support the growth of yeast mutants at both permissive and non-permissive temperature. Neither the full-length nor mature forms of chloroplast-located AtHsp90-5, mitochondria-located AtHsp90-6 and endoplasmic reticulum (ER)-located AtHsp90-7 could complement the yeast Hsp90 proteins. The cytosolic AtHsp90s could stabilize the biomembrane of the temperature-sensitive Hsp90 mutant strains under stress conditions, while the organellar AtHsp90s could not protect the biomembrane of the temperature-sensitive Hsp90 mutant strains. Yeast two-hybrid results showed that either pre-protein or mature forms of organellar AtHsp90s could interact with cofactors cpHsp70, Hsp70, Hsp70t-2, Cyp40, p23 and a substrate protein of NOS, while cytosolic AtHsp90s could not interact with them. These results suggest that organellar and cytosolic AtHsp90s possibly work through different molecular mechanisms in forming chaperone complexes and performing their functional roles.

Tamoxifen enhances the Hsp90 molecular chaperone ATPase activity.

BACKGROUND: Hsp90 is an essential molecular chaperone that is also a novel anti-cancer drug target. There is growing interest in developing new drugs that modulate Hsp90 activity. METHODOLOGY/PRINCIPAL FINDINGS: Using a virtual screening approach, 4-hydroxytamoxifen, the active metabolite of the anti-estrogen drug tamoxifen, was identified as a putative Hsp90 ligand. Surprisingly, while all drugs targeting Hsp90 inhibit the chaperone ATPase activity, it was found experimentally that 4-hydroxytamoxifen and tamoxifen enhance rather than inhibit Hsp90 ATPase. CONCLUSIONS/SIGNIFICANCE: Hence, tamoxifen and its metabolite are the first members of a new pharmacological class of Hsp90 activators.

Overexpression of AtHsp90.2, AtHsp90.5 and AtHsp90.7 in Arabidopsis thaliana enhances plant sensitivity to salt and drought stresses.

Three AtHsp90 isoforms, cytosolic AtHsp90.2, chloroplast-located AtHsp90.5, and endoplasmic reticulum (ER)-located AtHsp90.7, were characterized by constitutive overexpressing their genes in Arabidopsis thaliana. Both types of the transgenic plants overexpressing cytosolic and organellar AtHsp90s showed reduced tolerance to salt and drought stresses with lower germination rates and fresh weights, but improved tolerance to high concentration of Ca(2+) comparing with the wild type plants. Transcriptional analysis of ABA-responsive genes, RD29A, RD22 and KIN2 under salt and drought stresses, indicated that the induction expression of these genes was delayed by constitutive overexpression of cytosolic AtHsp90.2, but was hardly affected by that of organellar AtHsp90.5 and AtHsp90.7. These results implied that Arabidopsis different cellular compartments-located Hsp90s in Arabidopsis might be involved in abiotic stresses by different functional mechanisms, probably through ABA-dependent or Ca(2+) pathways, and proper homeostasis of Hsp90 was critical for cellular stress response and/or tolerance in plants.

Molecular chaperone Hsp90 stabilizes Pih1/Nop17 to maintain R2TP complex activity that regulates snoRNA accumulation.

Hsp90 is a highly conserved molecular chaperone that is involved in modulating a multitude of cellular processes. In this study, we identify a function for the chaperone in RNA processing and maintenance. This functionality of Hsp90 involves two recently identified interactors of the chaperone: Tah1 and Pih1/Nop17. Tah1 is a small protein containing tetratricopeptide repeats, whereas Pih1 is found to be an unstable protein. Tah1 and Pih1 bind to the essential helicases Rvb1 and Rvb2 to form the R2TP complex, which we demonstrate is required for the correct accumulation of box C/D small nucleolar ribonucleoproteins. Together with the Tah1 cofactor, Hsp90 functions to stabilize Pih1. As a consequence, the chaperone is shown to affect box C/D accumulation and maintenance, especially under stress conditions. Hsp90 and R2TP proteins are also involved in the proper accumulation of box H/ACA small nucleolar RNAs.

Expression of a constitutively activated plasma membrane H+-ATPase alters plant development and increases salt tolerance.

The plasma membrane proton pump ATPase (H(+)-ATPase) plays a major role in the activation of ion and nutrient transport and has been suggested to be involved in several physiological processes, such as cell expansion and salt tolerance. Its activity is regulated by a C-terminal autoinhibitory domain that can be displaced by phosphorylation and the binding of regulatory 14-3-3 proteins, resulting in an activated enzyme. To better understand the physiological consequence of this activation, we have analyzed transgenic tobacco (Nicotiana tabacum) plants expressing either wild-type plasma membrane H(+)-ATPase4 (wtPMA4) or a PMA4 mutant lacking the autoinhibitory domain (DeltaPMA4), generating a constitutively activated enzyme. Plants showing 4-fold higher expression of wtPMA4 than untransformed plants did not display any unusual phenotype and their leaf and root external acidification rates were not modified, while their in vitro H(+)-ATPase activity was markedly increased. This indicates that, in vivo, H(+)-ATPase overexpression is compensated by down-regulation of H(+)-ATPase activity. In contrast, plants that expressed DeltaPMA4 were characterized by a lower apoplastic and external root pH, abnormal leaf inclination, and twisted stems, suggesting alterations in cell expansion. This was confirmed by in vitro leaf extension and curling assays. These data therefore strongly support a direct role of H(+)-ATPase in plant development. The DeltaPMA4 plants also displayed increased salt tolerance during germination and seedling growth, supporting the hypothesis that H(+)-ATPase is involved in salt tolerance.

Molecular interaction network of the Hsp90 chaperone system.

Hsp90 is an essential and ubiquitous molecular chaperone that is required for the proper folding of a set of client proteins at a late stage in their folding process. In eukaryotes, cytoplasmic Hsp90 is absolutely essential for cell viability under all growth conditions. The functional cycle of the Hsp90 system requires a cohort of cochaperones and cofactors that regulate the activity of this chaperone. Hence, Hsp90 function is highly complex; in order to understand that complexity, several groups have attempted to map out the interaction network of this chaperone in yeast and mammalian systems using the latest available proteomic and genomic tools. Interaction networks emerging from these large scale efforts clearly demonstrate that Hsp90 plays a central role effecting multiple pathways and cellular processes. In yeast Saccharomyces cerevisiae, Hsp90 was shown to interact directly or indirectly with at least 10% of the yeast ORFs. The systematic application of large scale approaches to map out the Hsp90 chaperone network should allow the determination of the mechanisms employed by this chaperone system to maintain protein homeostasis in the cell.