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J. Biol. Chem., Vol. 279, Issue 34, 35692-35701, August 20, 2004

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Amino Acid Substitutions in the C-terminal AAA⁺ Module of Hsp104 Prevent Substrate Recognition by Disrupting Oligomerization and Cause High Temperature Inactivation^*

Johnny M. Tkach and John R. Glover

From the Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada

Hsp104 is an important determinant of thermotolerance in yeast and is an unusual molecular chaperone that specializes in the remodeling of aggregated proteins. The structural requirements for Hsp104-substrate interactions remain unclear. Upon mild heat shock Hsp104 formed cytosolic foci in live cells that indicated co-localization of the chaperone with aggregates of thermally denatured proteins. We generated random amino acid substitutions in the C-terminal 199 amino acid residues of a GFP-Hsp104 fusion protein, and we used a visual screen to identify mutants that remained diffusely distributed immediately after heat shock. Multiple amino acid substitutions were required for loss of heat-inducible redistribution, and this correlated with complete loss of nucleotide-dependent oligomerization. Based on the multiply substituted proteins, several single amino acid substitutions were generated by site-directed mutagenesis. The singly substituted proteins retained the ability to oligomerize and detect substrates. Intriguingly, some derivatives of Hsp104 functioned well in prion propagation and multiple stress tolerance but failed to protect yeast from extreme thermal stress. We demonstrate that these proteins co-aggregate in the presence of other thermolabile proteins during heat treatment both in vitro and in vivo suggesting a novel mechanism for uncoupling the function of Hsp104 in acute severe heat shock from its functions at moderate temperatures.

Received for publication, January 23, 2004 , and in revised form, April 27, 2004.

^* This work was supported in part by operating funds from the Canadian Institutes for Health Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a scholarship from the Natural Sciences and Engineering Research Council of Canada.

Supported by a scholarship from the Canadian Institutes for Health Research. To whom correspondence should be addressed: Dept. of Biochemistry, University of Toronto, Rm. 5302 Medical Sciences Bldg., 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada. Tel.: 416-978-3008; Fax: 416-978-8548; E-mail: john.glover{at}utoronto.ca.

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