Definition of the minimal fragments of Sti1 required for dimerization, interaction with Hsp70 and Hsp90 and in vivo functions

Gary Flom; Robert H Behal; Luke Rosen; Douglas G Cole; Jill L Johnson

doi:10.1042/BJ20070084

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Definition of the minimal fragments of Sti1 required for dimerization, interaction with Hsp70 and Hsp90 and in vivo functions

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Definition of the minimal fragments of Sti1 required for dimerization, interaction with Hsp70 and Hsp90 and in vivo functions

Gary Flom, Robert H Behal, Luke Rosen, Douglas G Cole and Jill L Johnson

Biochemical journal, Vol.404(Pt 1), pp.159-167

05/15/2007

DOI: https://doi.org/10.1042/BJ20070084

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https://hdl.handle.net/2376/115982

PMCID: PMC1868838

PMID: 17300223

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Abstract

Hsp, heat-shock protein

molecular chaperone

5-FOA, 5-fluororotic acid

WT, wild-type

GST, glutathione S-transferase

TPR, tetratricopeptide repeat

Hop

heat-shock protein

DP, aspartic acid–proline repeat

GR, glucocorticoid receptor

tetratricopeptide repeat domain

Hsc, heat-shock cognate protein

The molecular chaperone Hsp (heat-shock protein) 90 is critical for the activity of diverse cellular client proteins. In a current model, client proteins are transferred from Hsp70 to Hsp90 in a process mediated by the co-chaperone Sti1/Hop, which may simultaneously interact with Hsp70 and Hsp90 via separate TPR (tetratricopeptide repeat) domains, but the mechanism and in vivo importance of this function is unclear. In the present study, we used truncated forms of Sti1 to determine the minimal regions required for the Hsp70 and Hsp90 interaction, as well as Sti1 dimerization. We found that both TPR1 and TPR2B contribute to the Hsp70 interaction in vivo and that mutations in both TPR1 and TPR2B were required to disrupt the in vitro interaction of Sti1 with the C-terminus of the Hsp70 Ssa1. The TPR2A domain was required for the Hsp90 interaction in vivo , but the isolated TPR2A domain was not sufficient for the Hsp90 interaction unless combined with the TPR2B domain. However, isolated TPR2A was both necessary and sufficient for purified Sti1 to migrate as a dimer in solution. The DP2 domain, which is essential for in vivo function, was dispensable for the Hsp70 and Hsp90 interaction, as well as Sti1 dimerization. As evidence for the role of Sti1 in mediating the interaction between Hsp70 and Hsp90 in vivo , we identified Sti1 mutants that result in reduced recovery of Hsp70 in Hsp90 complexes. We also identified two Hsp90 mutants that exhibit a reduced Hsp70 interaction, which may help clarify the mechanism of client transfer between the two molecular chaperones.

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Title: Definition of the minimal fragments of Sti1 required for dimerization, interaction with Hsp70 and Hsp90 and in vivo functions
Creators: Gary Flom - Department of Microbiology, Molecular Biology and Biochemistry, and the Center for Reproductive Biology, University of Idaho, Moscow, ID 83844-3052, U.S.A
Robert H Behal - Department of Microbiology, Molecular Biology and Biochemistry, and the Center for Reproductive Biology, University of Idaho, Moscow, ID 83844-3052, U.S.A
Luke Rosen - Department of Microbiology, Molecular Biology and Biochemistry, and the Center for Reproductive Biology, University of Idaho, Moscow, ID 83844-3052, U.S.A
Douglas G Cole - Department of Microbiology, Molecular Biology and Biochemistry, and the Center for Reproductive Biology, University of Idaho, Moscow, ID 83844-3052, U.S.A
Jill L Johnson - Department of Microbiology, Molecular Biology and Biochemistry, and the Center for Reproductive Biology, University of Idaho, Moscow, ID 83844-3052, U.S.A
Publication Details: Biochemical journal, Vol.404(Pt 1), pp.159-167
Academic Unit: Center for Reproductive Biology
Publisher: Portland Press Ltd
Identifiers: 99900547618101842
Language: English
Resource Type: Journal article