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Eukaryotic Cell, May 2005, p. 849-860, Vol. 4, No. 5
1535-9778/05/$08.00+0     doi:10.1128/EC.4.5.849-860.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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)

Stefan H. Millson,1,{dagger} Andrew W. Truman,1,{dagger} Victoria King,1 Chrisostomos Prodromou,2 Laurence H. Pearl,2 and Peter W. Piper1*

Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN,1 Section for Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Rd., London SW3 6JB, United Kingdom2

Received 17 January 2005/ Accepted 22 February 2005

The 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 ~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.

* Corresponding author. Mailing address: Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, United Kingdom. Phone: (44) (0) 114 2222851. Fax: (44) (0) 114 222 2800. E-mail: peter.piper{at}sheffield.ac.uk.

{dagger} These authors made equal contributions to this study.

Eukaryotic Cell, May 2005, p. 849-860, Vol. 4, No. 5
1535-9778/05/$08.00+0     doi:10.1128/EC.4.5.849-860.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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