Saccharomyces cerevisiae Hsp104 protein. Purification and characterization of ATP-induced structural changes.
Heat-shock proteins (hsps) function in a variety of ways to help cells and organisms cope with environmental changes. One class of hsps, the Hsp100 proteins, is especially important for tolerance to a variety of extremely stressful conditions (e.g. high temperatures or high concentrations of ethanol). To begin to characterize the mechanism of action of Hsp100 proteins, we have initiated an in vitro analysis of the Saccharomyces cerevisiae Hsp104 protein. Here, we report the purification and initial structural characterization of the wild-type protein and three variants carrying mutations in the two ATP-binding site consensus elements. As demonstrated by both gel filtration chromatography and by cross-linking studies with glutaraldehyde, Hsp104 forms a homohexameric particle. By electron microscopy, these particles are ring-shaped and reminiscent of proteins in the Hsp60 and TF55/TCP families. In contrast to these other proteins, Hsp104 forms single rings, each containing only six subunits. More strikingly, the assembly and maintenance of Hsp104 particles are dependent upon the presence of adenine nucleotides. Oligomerization appears to primarily depend upon the second of the two ATP-binding sites in the protein.
Parsell
D A
DA
Department of Molecular Genetics and Cell Biology, University of Chicago, Illinois 60637.
Kowal
A S
AS
Lindquist
S
S
D000255:Adenosine Triphosphate / Q000494:pharmacology; D003432:Cross-Linking Reagents; D005656:Fungal Proteins / Q000302:isolation & purification; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000302:isolation & purification; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008970:Molecular Weight; D010449:Peptide Mapping; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000737:chemistry*
D003432:Cross-Linking Reagents; D005656:Fungal Proteins; D006360:Heat-Shock Proteins; D046911:Macromolecular Substances; D000255:Adenosine Triphosphate