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.

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.

The plasma membrane proton pump ATPase (H 1 -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 1 -ATPase4 (wtPMA4) or a PMA4 mutant lacking the autoinhibitory domain (DPMA4), 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 1 -ATPase activity was markedly increased. This indicates that, in vivo, H 1 -ATPase overexpression is compensated by down-regulation of H 1 -ATPase activity. In contrast, plants that expressed DPMA4 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 1 -ATPase in plant development. The DPMA4 plants also displayed increased salt tolerance during germination and seedling growth, supporting the hypothesis that H 1 -ATPase is involved in salt tolerance.The H 1 -ATPase transports protons out of the cell across the plasma membrane, thus establishing the proton electrochemical gradient that contributes to the maintenance of the intracellular and extracellular pHs and drives secondary transport of ions and metabolites. As solute transport is directly related to osmotic water movement, the H 1

Molecular chaperones are essential components of a quality control machinery present in the cell. They can either aid in the folding and maintenance of newly translated proteins, or they can lead to the degradation of misfolded and destabilized proteins. Hsp90 is a key member of this machinery. It is a ubiquitous molecular chaperone that is found in eubacteria and all branches of eukarya. It plays a central role in cellular signaling since it is essential for maintaining the activity of several signaling proteins, including steroid hormone receptors and protein kinases. Hsp90 is currently a novel anticancer drug target since it is overexpressed in some cancer cells. The chaperone typically functions as part of large complexes, which include other chaperones and essential cofactors that regulate its function. It is thought that different cofactors target Hsp90 to different sets of substrates. However, the mechanism of Hsp90 function remains poorly understood. As part of an effort to elucidate the Hsp90 chaperone network, we carried out a large-scale proteomics study to identify physical and genetic interactors of the chaperone. We identified 2 highly conserved novel Hsp90 cofactors, termed Tah1 and Pih1, that bind to the chaperone and that also associate physically and functionally with the essential DNA helicases Rvb1 and Rvb2. These helicases are key components of the chromatin remodeling complexes Ino80 and SWR-C. Tah1 and Pih1 seem to represent a novel class of Hsp90 cofactors that allow the chaperone to indirectly affect gene regulation in the cell in addition to its ability to directly promote protein folding. In this review, we provide an overview of Hsp90 structure and function, and we discuss the literature that links the chaperone activity to gene regulation.