Abstract
Hsp90 is an essential eukaryotic chaperone with a role in folding specific “client” proteins such as kinases and hormone receptors. Previously performed homozygous diploid yeast deletion collection screens uncovered broad requirements for Hsp90 in cellular transport and cell cycle progression. These screens also revealed that the requisite cellular functions of Hsp90 change with growth temperature. We present here for the first time the results of heterozygous deletion collection screens conducted at the hypothermic stress temperature of 15°C. Extensive bioinformatic analyses were performed on the resulting data in combination with data from homozygous and heterozygous screens previously conducted at normal (30°C) and hyperthermic stress (37°C) growth temperatures. Our resulting meta-analysis uncovered extensive connections between Hsp90 and (1) general transcription, (2) ribosome biogenesis and (3) GTP binding proteins. Predictions from bioinformatic analyses were tested experimentally, supporting a role for Hsp90 in ribosome stability. Importantly, the integrated analysis of the 15°C heterozygous deletion pool screen with previously conducted 30°C and 37°C screens allows for essential genetic targets of Hsp90 to emerge. Altogether, these novel contributions enable a more complete picture of essential Hsp90 functions.
Highlights
Hsp90 is an essential and abundant eukaryotic chaperone with an established role in the folding of ‘‘client’’ proteins involved in cellular signaling pathways, primarily kinases and steroid hormone receptors
We conduct a meta-analysis of all homozygous and heterozygous deletion pool screens, along with combined analysis of all curated Hsp90 interactors, permitting us to build a complete picture of Hsp90 function in yeast. These analyses critically examine unnamed ORFs identified in these screens, identify novel essential cellular targets that may underlie the necessity of Hsp90 for viability, identify critical hubs of Hsp90 action in cellular processes, and allow genome-wide predictions of bona fide Hsp90 substrates and chaperones, all of which further contribute to our knowledge of what governs Hsp90-substrate and Hsp90-cochaperone interactions
We showed that the top 5% homozygous deletion strains most sensitive to Hsp90 inhibition were significantly enriched for established Hsp90 interactors, validating their potential to reveal novel Hsp90 substrates and cochaperones [40]
Summary
Hsp is an essential and abundant eukaryotic chaperone with an established role in the folding of ‘‘client’’ proteins involved in cellular signaling pathways, primarily kinases and steroid hormone receptors. Hsp has been shown to participate in the cytosolic quality control of proteins that are not its folding substrates [9] These connections between Hsp and quality control, including its ability in vitro to prevent protein aggregation [10], as well as its affiliation with Aß, tau, and alpha-synuclein aggregates [11,12,13,14], suggest that Hsp function may be a critical determinant in the onset or severity of neurodegenerative diseases linked to protein misfolding [15]. As recent structural and biochemical studies have demonstrated, the transient ATPdependent dimerization of Hsp N-termini appears to be conserved in the non-essential bacterial homologue HtpG [18], yeast Hsp90 [19,20], and human Hsp90ß [21,22], suggesting a conserved ATPase cycling mechanism involving dramatic conformational rearrangements
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