Eukaryotic_Cell-2005-Millson-849-60.pdf (326.18 kB)
A two-hybrid screen of the yeast proteome for Hsp90 interactors uncovers a novel Hsp90 chaperone requirement in the activity of a stress-activated mitogen-activated protein kinase, Slt2p (Mpk1p)
journal contribution
posted on 2023-06-08, 14:42 authored by Stefan H Millson, Andrew W Truman, Victoria King, Chrisostomos ProdromouChrisostomos Prodromou, Laurence PearlLaurence Pearl, Peter W PiperThe Hsp90 chaperone cycle catalyzes the final activation step of several important eukaryotic proteins (Hsp90 "clients"). Although largely a functional form of Hsp90, an Hsp90-Gal4p DNA binding domain fusion (Hsp90-BD) displays no strong interactions in the yeast two-hybrid system, consistent with a general transience of most Hsp90-client associations. Strong in vivo interactions are though detected when the E33A mutation is introduced into this bait, a mutation that should arrest Hsp90-client complexes at a stage where the client is stabilized, yet prevented from attaining its active form. This E33A mutation stabilized the two-hybrid interactions of the Hsp90-BD fusion with approximately 3% of the Saccharomyces cerevisiae proteome in a screen of the 6,000 yeast proteins expressed as fusions to the Gal4p activation domain (AD). Among the detected interactors were the two stress-activated mitogen-activated protein (MAP) kinases of yeast, Hog1p and Slt2p (Mpk1p). Column retention experiments using wild-type and mutant forms of Hsp90 and Slt2p MAP kinase, as well as quantitative measurements of the effects of stress on the two-hybrid interaction of mutant Hsp90-BD and AD-Slt2p fusions, revealed that Hsp90 binds exclusively to the dually Thr/Tyr-phosphorylated, stress-activated form of Slt2p [(Y-P,T-P)Slt2p] and also to the MAP kinase domain within this (Y-P,T-P)Slt2p. Phenotypic analysis of a yeast mutant that expresses a mutant Hsp90 (T22Ihsp82) revealed that Hsp90 function is essential for this (Y-P,T-P)Slt2p to activate one of its downstream targets, the Rlm1p transcription factor. The interaction between Hsp90 and (Y-P,T-P)Slt2p, characterized in this study, is probably essential in this Hsp90 facilitation of the Rlm1p activation by Slt2p.
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Publication status
- Published
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- Published version
Journal
Eukaryotic CellISSN
1535-9778Publisher
American Society for MicrobiologyExternal DOI
Issue
5Volume
4Page range
849-860Department affiliated with
- Biochemistry Publications
Full text available
- Yes
Peer reviewed?
- Yes
Legacy Posted Date
2015-02-25First Open Access (FOA) Date
2015-02-25First Compliant Deposit (FCD) Date
2015-02-25Usage metrics
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No categories selectedKeywords
Caffeine/pharmacologyHSP90 Heat-Shock Proteins/genetics/*metabolismMitogen-Activated Protein Kinases/*metabolismMutationPhosphorylationProteome/genetics/*metabolismSaccharomyces cerevisiae/drug effects/genetics/*metabolismSaccharomyces cerevisiae Proteins/genetics/*metabolismTwo-Hybrid System Techniques
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