Summary In parallel with evolutionary developments, the Hsp90 molecular chaperone system shifted from a simple prokaryotic factor into an expansive network that includes a variety of cochaperones. We have taken high-throughput genomic and proteomic approaches to better understand the abundant yeast p23 cochaperone Sba1. Our work revealed an unexpected p23 network that displayed considerable independence from known Hsp90 clients. Additionally, our data uncovered a broad nuclear role for p23, contrasting with the historical dogma of restricted cytosolic activities for molecular chaperones. Validation studies demonstrated that yeast p23 was required for proper Golgi function, ribosome biogenesis and was necessary for efficient DNA repair from a wide range of mutagens. Notably, mammalian p23 had conserved roles in these pathways as well as being necessary for proper cell mobility. Taken together, our work demonstrates that the p23 chaperone serves a broad physiological network and functions both in conjunction with and sovereign to Hsp90.

Distinct protein assemblies are nucleated at telomeric DNA to both guard the ends from damage and lengthen the DNA following replication. In yeast, Cdc13 recruits either Stn1/Ten1 to form a protective cap or the telomerase holoenzyme to extend the DNA. We have established an in vitro yeast telomere system in which Stn1/Ten1-unextendable or telomerase-extendable states can be observed. Both assemblies are Cdc13-dependent, as the Cdc13 C-terminal region supports Stn1/Ten1 interactions and the N-terminal region contains a telomerase activation function. Notably, the yeast Hsp90 chaperone Hsp82 mediates the switch between the telomere capping and extending structures by modulating the DNA binding activity of Cdc13. Taken together, our data demonstrate that the Hsp82 chaperone facilitates telomere DNA maintenance by promoting transitions between different operative complexes and by reducing the potential for binding events that would otherwise block the assembly of downstream structures.

Enterohemorrhagic Escherichia coli (EHEC) is a water- and food-borne pathogen that causes hemorrhagic colitis. EHEC uses a type III secretion system (T3SS) to translocate effector proteins that subvert host cell function. T3SS-substrates encoded outside of the locus of enterocyte effacement are important to E. coli pathogenesis. We discovered an EHEC secreted protein, NleF, encoded by z6020 in O-island 71 of E. coli EDL933 that we hypothesized to be a T3SS substrate. Experiments are presented that probe the function of NleF and its role in virulence. Immunoblotting of secreted and translocated proteins suggest that NleF is secreted by the T3SS and is translocated into host cells in vitro where it localizes to the host cytoplasm. Infection of HeLa cells with E. coli possessing or lacking nleF and transient expression of NleF-GFP via transfection did not reveal a significant role for NleF in several assays of bacterial adherence, host cytoskeletal remodeling, or host protein secretion. However, competitive coinfection of mice with Citrobacter rodentium strains possessing or lacking nleF suggested a contribution of NleF to bacterial colonization. Challenge of gnotobiotic piglets also revealed a role for NleF in colonization of the piglet colon and rectoanal junction.

Cellular environments are highly complex and contain a copious variety of proteins that must operate in unison to achieve homeostasis. To guide and preserve order, multifaceted molecular chaperone networks are present within each cell type. To handle the vast client diversity and regulatory demands, a wide assortment of chaperones are needed. In addition to the classic heat shock proteins, cochaperones with inherent chaperoning abilities (e.g., p23, Hsp40, Cdc37, etc.) are likely used to complete a system. In this review, we focus on the HSP90-associated cochaperones and provide evidence supporting a model in which select cochaperones are used to differentially modulate target proteins, contribute to combinatorial client regulation, and increase the overall reach of a cellular molecular chaperone network. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).

Molecular chaperones govern protein homeostasis, being allied to the beginning (folding) and ending (degradation) of the protein life cycle. Yet, the Hsp90 system primarily associates with native factors, including fully assembled complexes. The significance of these connections is poorly understood. To delineate why Hsp90 and its cochaperone p23 interact with a mature structure, we focused on the RSC chromatin remodeler. Both Hsp90 and p23 triggered the release of RSC from DNA or a nucleosome. Although Hsp90 only freed bound RSC, p23 enhanced nucleosome remodeling prior to discharging the complex. In vivo, RSC mobility and remodeling function were chaperone dependent. Our results suggest Hsp90 and p23 contribute to proteostasis by chaperoning mature factors through energetically unfavorable events, thereby maintaining the cellular pool of active native proteins. In the case of RSC, p23 and Hsp90 promote a dynamic action, allowing a limited number of remodelers to effectively maintain chromatin in a pliable state.