The Hsp90-Dependent Proteome Is Conserved and Enriched for Hub Proteins with High Levels of Protein–Protein Connectivity

Abstract

Hsp90 is one of the most abundant and conserved proteins in the cell. Reduced levels or activity of Hsp90 causes defects in many cellular processes and also reveals genetic and nongenetic variation within a population. Despite information about Hsp90 protein–protein interactions, a global view of the Hsp90-regulated proteome in yeast is unavailable. To investigate the degree of dependency of individual yeast proteins on Hsp90, we used the “stable isotope labeling by amino acids in cell culture” method coupled with mass spectrometry to quantify around 4,000 proteins in low-Hsp90 cells. We observed that 904 proteins changed in their abundance by more than 1.5-fold. When compared with the transcriptome of the same population of cells, two-thirds of the misregulated proteins were observed to be affected posttranscriptionally, of which the majority were downregulated. Further analyses indicated that the downregulated proteins are highly conserved and assume central roles in cellular networks with a high number of protein interacting partners, suggesting that Hsp90 buffers genetic and nongenetic variation through regulating protein network hubs. The downregulated proteins were enriched for essential proteins previously not known to be Hsp90-dependent. Finally, we observed that downregulation of transcription factors and mating pathway components by attenuating Hsp90 function led to decreased target gene expression and pheromone response, respectively, providing a direct link between observed proteome regulation and cellular phenotypes.

Responding to a Zoonotic Emergency with Multi-omics Research:Pentatrichomonas hominisHydrogenosomal Protein Characterization with Use of RNA Sequencing and Proteomics

“…The sample preparation for mass spectrometry (MS) was performed as previously described (Gopinath et al, 2014). Briefly, 5 lg hydrogenosome extract was dissolved in 50 mM NH 4 HCO 3 containing 0.1% SDS, followed by 5 mM DTT at 56°C for 30 min, 15 mM iodoacetamide at RT for 30 min, and digested with 0.5 lg trypsin at 37°C overnight.…”

Hsp90 Maintains Proteostasis of the Galactose Utilization Pathway To Prevent Cell Lethality

“…Our study revealed this phenomenon using the GAL system, which had been overlooked by studies conducted earlier because of their focus on glucose-rich media. Nevertheless, a recent proteomic study has also revealed that many proteins whose abundance is influenced by the level of Hsp90 are not known Hsp90 clients, and many of them belong to the same pathways (18). A thorough understanding of how Hsp90 influences the GAL pathway will allow us to further elucidate the complexity of the Hsp90 network.…”

“…However, in sugar metabolism, the induction of genes constitutes only a part of the process. It is equally important to investigate how the protein products of those genes are regulated after induction (18,19,27). Here, we report an atypical mechanism by which Hsp90 affects the galactose utilization pathway, involving direct maintenance of both structural and regulatory Gal proteins after galactose induction.…”

“…A majority of protein chaperones are heat shock proteins (HSPs) that are induced by stress conditions and typically bind to numerous other proteins (13)(14)(15). Among these HSPs, Hsp90 is unique as it assists very specific substrates known as "clients" at a late stage of folding, and these client proteins are broadly involved in signal transduction and gene regulation (16)(17)(18). Hsp90 is a dimeric ATPase required for cell viability even under normal conditions (19).…”

“…Several studies point out that Hsp90 usually influences the stability of these key master regulators and, therefore, has strong impacts on their entire pathways (19). For example, Hsp90 affects signal transduction of the Saccharomyces cerevisiae mating pathway, so that inhibition or mutation of Hsp90 results in downregulation of the components of the mitogen-activated protein kinase (MAPK) cascade and leads to reduced mating ability (18,27).…”