ABSTRACT
The Hsp90 chaperone machinery in eukaryotes comprises a number of distinct accessory factors. Cns1 is one of the few essential co-chaperones in yeast, but its structure and function remained unknown. Here, we report the X-ray structure of the Cns1 fold and NMR studies on the partly disordered, essential segment of the protein. We demonstrate that Cns1 is important for maintaining translation elongation, specifically chaperoning the elongation factor eEF2. In this context, Cns1 interacts with the novel co-factor Hgh1 and forms a quaternary complex together with eEF2 and Hsp90. The in vivo folding and solubility of eEF2 depend on the presence of these proteins. Chaperoning of eEF2 by Cns1 is essential for yeast viability and requires a defined subset of the Hsp90 machinery as well as the identified eEF2 recruiting factor Hgh1.
Subject(s)
HSP90 Heat-Shock Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Molecular Chaperones/metabolism , Peptide Chain Elongation, Translational , Peptide Elongation Factor 2/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Crystallography, X-Ray , Peptidyl-Prolyl Isomerase F , Cyclophilins/genetics , Cyclophilins/metabolism , HSP90 Heat-Shock Proteins/chemistry , HSP90 Heat-Shock Proteins/genetics , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/genetics , Models, Molecular , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Nuclear Magnetic Resonance, Biomolecular , Peptide Elongation Factor 2/chemistry , Peptide Elongation Factor 2/genetics , Protein Binding , Protein Folding , Protein Interaction Domains and Motifs , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Structure-Activity RelationshipABSTRACT
The heat shock protein 90 (HSP90) chaperone machinery is a key regulator of proteostasis under both physiological and stress conditions in eukaryotic cells. As HSP90 has several hundred protein substrates (or 'clients'), it is involved in many cellular processes beyond protein folding, which include DNA repair, development, the immune response and neurodegenerative disease. A large number of co-chaperones interact with HSP90 and regulate the ATPase-associated conformational changes of the HSP90 dimer that occur during the processing of clients. Recent progress has allowed the interactions of clients with HSP90 and its co-chaperones to be defined. Owing to the importance of HSP90 in the regulation of many cellular proteins, it has become a promising drug target for the treatment of several diseases, which include cancer and diseases associated with protein misfolding.
Subject(s)
HSP90 Heat-Shock Proteins/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Animals , HSP90 Heat-Shock Proteins/chemistry , HSP90 Heat-Shock Proteins/genetics , Humans , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Protein Binding , Protein FoldingABSTRACT
Hsp90 couples ATP hydrolysis to large conformational changes essential for activation of client proteins. The structural transitions involve dimerization of the N-terminal domains and formation of 'closed states' involving the N-terminal and middle domains. Here, we used Hsp90 mutants that modulate ATPase activity and biological function as probes to address the importance of conformational cycling for Hsp90 activity. We found no correlation between the speed of ATP turnover and the in vivo activity of Hsp90: some mutants with almost normal ATPase activity were lethal, and some mutants with lower or undetectable ATPase activity were viable. Our analysis showed that it is crucial for Hsp90 to attain and spend time in certain conformational states: a certain dwell time in open states is required for optimal processing of client proteins, whereas a prolonged population of closed states has negative effects. Thus, the timing of conformational transitions is crucial for Hsp90 function and not cycle speed.
Subject(s)
Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , HSP90 Heat-Shock Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , HSP90 Heat-Shock Proteins/chemistry , HSP90 Heat-Shock Proteins/genetics , Models, Molecular , Point Mutation , Protein Conformation , Protein Domains , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Membrane phospholipids typically contain fatty acids (FAs) of 16 and 18 carbon atoms. This particular chain length is evolutionarily highly conserved and presumably provides maximum stability and dynamic properties to biological membranes in response to nutritional or environmental cues. Here, we show that the relative proportion of C16 versus C18 FAs is regulated by the activity of acetyl-CoA carboxylase (Acc1), the first and rate-limiting enzyme of FA de novo synthesis. Acc1 activity is attenuated by AMPK/Snf1-dependent phosphorylation, which is required to maintain an appropriate acyl-chain length distribution. Moreover, we find that the transcriptional repressor Opi1 preferentially binds to C16 over C18 phosphatidic acid (PA) species: thus, C16-chain containing PA sequesters Opi1 more effectively to the ER, enabling AMPK/Snf1 control of PA acyl-chain length to determine the degree of derepression of Opi1 target genes. These findings reveal an unexpected regulatory link between the major energy-sensing kinase, membrane lipid composition, and transcription.
