title: Amino acid substitutions in the C-terminal AAA+ module of Hsp104 prevent substrate recognition by disrupting oligomerization and cause high temperature inactivation.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Johnny M
authors.initials: JM
authors.lastname: Tkach
authors.forename: John R
authors.initials: JR
authors.lastname: Glover
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D019943:Amino Acid Substitution; D019281:Dimerization; D004789:Enzyme Activation; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D006360:Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C070972:HsP104 protein, S cerevisiae; D000251:Adenosine Triphosphatases