Rongmin Zhao

Navigating the Chaperone Network: An Integrative Map of Physical and Genetic Interactions Mediated by the Hsp90 Chaperone

Physical, genetic, and chemical-genetic interactions centered on the conserved chaperone Hsp90 were mapped at high resolution in yeast using systematic proteomic and genomic methods. Physical interactions were identified using genome-wide two hybrid screens combined with large-scale affinity purification of Hsp90-containing protein complexes. Genetic interactions were uncovered using synthetic genetic array technology and by a microarray-based chemical-genetic screen of a set of about 4700 viable yeast gene deletion mutants for hypersensitivity to the Hsp90 inhibitor geldanamycin. An extended network, consisting of 198 putative physical interactions and 451 putative genetic and chemical-genetic interactions, was found to connect Hsp90 to cofactors and substrates involved in a wide range of cellular functions. Two novel Hsp90 cofactors, Tah1 (YCR060W) and Pih1 (YHR034C), were also identified. These cofactors interact physically and functionally with the conserved AAA(+)-type DNA helicases Rvb1/Rvb2, which are key components of several chromatin remodeling factors, thereby linking Hsp90 to epigenetic gene regulation.

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 (ΔPMA4), 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 ΔPMA4 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 ΔPMA4 plants also displayed increased salt tolerance during germination and seedling growth, supporting the hypothesis that H+-ATPase is involved in salt tolerance.

Cosuppression of a Plasma Membrane H + -ATPase Isoform Impairs Sucrose Translocation, Stomatal Opening, Plant Growth, and Male Fertility

The plasma membrane H+-ATPase builds up a pH and potential gradient across the plasma membrane, thus activating a series of secondary ion and metabolite transporters. pma4 (for plasma membrane H+-ATPase 4), the most widely expressed H+-ATPase isogene in Nicotiana plumbaginifolia, was overexpressed in tobacco. Plants that overexpressed PMA4 showed no major changes in plant growth under normal conditions. However, two transformants were identified by their stunted growth, slow leaf initiation, delayed stem bolting and flowering, and male sterility. Protein gel blot analysis showed that expression of the endogenous and transgenic pma4 was cosuppressed. Cosuppression was developmentally regulated because PMA4 was still present in developing leaves but was not detected in mature leaves. The glucose and fructose content increased threefold, whereas the sucrose content remained unchanged. The rate of sucrose exudation from mature leaves was reduced threefold and the sugar content of apical buds was reduced twofold, suggesting failure of sucrose loading and translocation to the sink tissues. Cosuppression of PMA4 also affected the guard cells, stomatal opening, and photosynthesis in mature leaves. These results show that a single H+-ATPase isoform plays a major role in several transport-dependent physiological processes.

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.

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 Arabidopsisthaliana. 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 Ca2+ 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 Ca2+ pathways, and proper homeostasis of Hsp90 was critical for cellular stress response and/or tolerance in plants.

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

Background: Tah1 and Pih1 are Hsp90 interactors that form a ternary complex with the chaperone. Results: NMR structure of Tah1 revealed the presence of two tetratricopeptide repeat motifs followed by a C helix and an unstructured region. Conclusion: Tah1 can bind simultaneously two other proteins using different interaction modes. Significance: The study provides important insights into protein complex assembly. 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 analysis of the plasma membrane H+-ATPase pma4 transcription promoter from Nicotiana plumbaginifolia activated by the CaMV 35S promoter enhancer

pma4 is the major plasma membrane H+-ATPase gene in Nicotiana plumbaginifolia. To study its physiological role by overexpression, we evaluated the possibility of enhancing the pma4 transcription promoter using a 165-bp enhancing sequence of the CaMV 35S transcription promoter. This was inserted into the pma4 promoter either 500 or 50 nucleotides upstream from the transcription start site. Transient expression with the gusA reporter gene showed that both enhanced pma4 promoters had a 4- to 13-fold greater transcription activity than the native pma4 promoter. Quantitative analysis of stable transgenic plants also showed that both enhanced pma4 promoters conferred much greater GUS activity. Histochemical assay showed that the enhanced promoters produced strong GUS activity in most cell types as already observed in the 35S or the native pma4 promoter. In the cell types where either the 35S or pma4 promoter was inactive, the enhanced promoters mimicked expression of the active one. However, there were cases (e.g. root cortex of seedlings) where, although both 35S and pma4 promoters were active, none of the enhanced promoters induced GUS activity. This might indicate the interference of promoter regulatory elements. The two enhanced pma4 promoters conferred similar expression throughout the plant development, implying that there was no regulatory element at either the pma4 -500 or -50 position, that conferred important tissue specificity

Specificity in substrate and cofactor recognition by the N-terminal domain of the chaperone ClpX

Clp ATPases are a unique group of ATP-dependent chaperones supporting targeted protein unfolding and degradation in concert with their respective proteases. ClpX is a representative member of these ATPases; it consists of two domains, a zinc-binding domain (ZBD) that forms dimers and a AAA+ ATP-binding domain that arranges into a hexamer. Analysis of the binding preferences of these two domains in ClpX revealed that both domains preferentially bind to hydrophobic residues but have different sequence preferences, with the AAA+ domain preferentially recognizing a wider range of specific sequences than ZBD. As part of this analysis, the binding site of the ClpX dimeric cofactor, SspB2, on ZBD in ClpX was determined by NMR and mutational analysis. The SspB C terminus was found to interact with a hydrophobic patch on the surface of ZBD. The affinity of SspB2 toward ZBD2 and the geometry of the SspB2–ZBD2 complex were investigated by using the newly developed quantitative optical biosensor method of dual polarization interferometry. The data suggest a model for the interaction between SspB2 and the ClpX hexamer.

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

Background: Pih1 is an unstable protein and forms an R2TP complex with Rvb1, Rvb2, and Tah1. Results: Pih1 contains two intrinsically disordered regions that mediate different protein-protein interactions within R2TP complex. Conclusion: Pih1 contains an N-terminal Rvb1/Rvb2-binding domain and a C-terminal regulatory domain. Significance: The study provides important insights into the mechanism of intrinsically disordered proteins in protein complex formation. 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, Pih11–230, was found to be able to complement the physiological role of full-length Pih1 at 37 °C. Pih11–230 as well as a shorter Pih1 N-terminal fragment Pih11–195 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.