Display Settings:

Format

Send to:

Choose Destination
We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Eukaryot Cell. 2005 May;4(5):849-60.

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).

Author information

  • 1Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, United Kingdom.

Abstract

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 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.

PMID:
15879519
[PubMed - indexed for MEDLINE]
PMCID:
PMC1140089
Free PMC Article

Images from this publication.See all images (5)Free text

FIG. 1.
FIG. 2.
FIG. 3.
FIG. 4.
FIG. 5.
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for HighWire Icon for PubMed Central
    Loading ...
    Write to the Help Desk