Subunit Interactions Influence the Biochemical and Biological Properties of Hsp104

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 98; no. 3; pp. 914 - 919
• Main Authors: Schirmer, Eric C., Ware, Danielle M., Queitsch, Christine, Kowal, Anthony S., Lindquist, Susan L.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 30.01.2001; National Acad Sciences; The National Academy of Sciences
• Subjects: Adenosine triphosphatases; Adenosine Triphosphatases - chemistry; Adenosine Triphosphatases - metabolism; Adenosine Triphosphate - metabolism; Amino Acid Substitution; Binding Sites; Biological Sciences; Coefficients; Computer software; Cooperation; Estradiol - pharmacology; Fungal Proteins - chemistry; Fungal Proteins - metabolism; Heat tolerance; Heat-Shock Proteins - chemistry; Heat-Shock Proteins - metabolism; Hsp104 protein; Hydrolysis; Kinetics; Mutagenesis, Site-Directed; Oligomers; Phosphates; Point Mutation; Protein Subunits; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Saccharomyces cerevisiae - drug effects; Saccharomyces cerevisiae - growth & development; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.031568098

Point mutations in either of the two nucleotide-binding domains (NBD) of Hsp104 (NBD1 and NBD2) eliminate its thermotolerance function in vivo. In vitro, NBD1 mutations virtually eliminate ATP hydrolysis with little effect on hexamerization; analogous NBD2 mutations reduce ATPase activity and severely impair hexamerization. We report that high protein concentrations overcome the assembly defects of NBD2 mutants and increase ATP hydrolysis severalfold, changing Vmaxwith little effect on Km. In a complementary fashion, the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate inhibits hexamerization of wild-type (WT) Hsp104, lowering Vmaxwith little effect on Km. ATP hydrolysis exhibits a Hill coefficient between 1.5 and 2, indicating that it is influenced by cooperative subunit interactions. To further analyze the effects of subunit interactions on Hsp104, we assessed the effects of mutant Hsp104 proteins on WT Hsp104 activities. An NBD1 mutant that hexamerizes but does not hydrolyze ATP reduces the ATPase activity of WT Hsp104 in vitro. In vivo, this mutant is not toxic but specifically inhibits the thermotolerance function of WT Hsp104. Thus, interactions between subunits influence the ATPase activity of Hsp104, play a vital role in its biological functions, and provide a mechanism for conditionally inactivating Hsp104 function in vivo.