The two major molecular chaperone families that mediate ATP-dependent protein folding and refolding are the heat shock proteins Hsp6Os (GroEL) and Hsp7Os (DnaK). Cip proteins, like chaperones, are highly conserved, present in all organisms, and contain ATP and polypeptide binding sites. We discovered that CipA, the ATPase component of the ATP-dependent ClpAP protease, is a molecular chaperone. CipA performs the ATP-dependent chaperone function of DnaK and DnaJ in the in vitro activation of the plasmid P1 RepA replication initiator protein. RepA is activated by the conversion of dimers to monomers. We show that CipA targets RepA for degradation by ClpP, demonstrating a direct link between the protein unfolding function of chaperones and proteolysis. In another chaperone assay, ClpA protects luciferase from irreversible heat inactivation but is unable to reactivate luciferase.Molecular chaperones interact with other proteins to mediate ATP-dependent protein folding, refolding, assembly, and disassembly of proteins. The Hsp7O chaperone system of Escherichia coli consists of the DnaK, DnaJ, and GrpE heat shock proteins. In vivo these three heat shock proteins function together in many cellular processes, as demonstrated by the observations that mutants in dnaK, dnaJ, and grpE have similar effects on DNA replication of E. coli, plasmids P1 and F and phage A, RNA synthesis, cell division, protein transport, regulation of the heat shock response, protection of enzymes from misfolding or aggregation during heat shock, and degradation of abnormal proteins (reviewed in ref.

-RssB-ClpXP complex forms. The complex degrades S and releases RssB from ClpXP in an ATP-dependent reaction. Our results illuminate an important mechanism for regulated protein turnover in which a unique targeting protein, whose own activity is regulated through specific signaling pathways, catalyzes the delivery of a specific substrate to a specific protease.

FtsZ is the major cytoskeletal protein in bacteria and a tubulin homologue. It polymerizes and forms a ring where constriction occurs to divide the cell. We found that FtsZ is degraded by E. coli ClpXP, an ATP-dependent protease. In vitro, ClpXP degrades both FtsZ protomers and polymers; however, polymerized FtsZ is degraded more rapidly than the monomer. Deletion analysis shows that the N-terminal domain of ClpX is important for polymer recognition and that the FtsZ C terminus contains a ClpX recognition signal. In vivo, FtsZ is turned over slower in a clpX deletion mutant compared with a WT strain. Overexpression of ClpXP results in increased FtsZ degradation and filamentation of cells. These results suggest that ClpXP may participate in cell division by modulating the equilibrium between free and polymeric FtsZ via degradation of FtsZ filaments and protomers.AAAϩ ATPase ͉ cell division ͉ ClpP ͉ proteolysis ͉ septum

Two members of the AAA+ superfamily, ClpB and Hsp104, collaborate with Hsp70 and Hsp40 to rescue aggregated proteins. However, the mechanisms that elicit and underlie their proteinremodeling activities remain unclear. We report that for both Hsp104 and ClpB, mixtures of ATP and ATPγS unexpectedly unleash activation, disaggregation, and unfolding activities independent of co-chaperones. Mutations reveal how remodeling activities are elicited by impaired hydrolysis at individual nucleotide binding domains. However, for some substrates, mixtures of ATP and ATPγS abolish remodeling, while for others ATP binding without hydrolysis is sufficient. Remodeling of different substrates necessitates a diverse balance of polypeptide holding (which requires ATP binding but not hydrolysis) and unfolding (which requires ATP hydrolysis). We suggest that this versatility in reaction mechanism enables ClpB and Hsp104 to reactivate the entire aggregated proteome after stress, and enables Hsp104 to control prion inheritance.Life demands that members of the AAA+ ATPase superfamily (ATPases associated with various cellular activities) couple energy from ATP hydrolysis to the remodeling of a bewildering array of macromolecular structures, that range from protein to DNA and RNA 1, 2 . Typically, eukaryotic genomes encode 50-80 family members 1 , each of which occupies specific niches that require specialized modes of substrate selection and regulation. The extraordinary adaptive radiation of AAA+ proteins to function in a multitude of cellular reactions illustrates the versatility of their structurally conserved AAA+ domain. Subunits containing AAA+ domains assemble into oligomeric rings, and ATP binds at the interface between adjacent protomers 1, 2 . AAA+ oligomers undergo considerable conformational changes during ATP binding and hydrolysis, although how these events are regulated and transduced into productive substrate remodeling remains largely enigmatic. Furthermore, it remains unanswered whether individual AAA+ family members rely on a common reaction mechanism to remodel various macromolecular clients. It is also unclear whether different AAA+ members have evolved distinct methods to engage and restructure substrates, or if individual proteins can switch between distinct reaction mechanisms for different substrates.Two members of the AAA+ superfamily separated by ~2 billion years of evolution 3 , yeast Hsp104, and its E. coli homolog, ClpB, allow cell survival after exposure to extreme environmental stress 4-7 . They function to dissolve and renature thousands of diverse 5 Correspondence: Sue Wickner,

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Yeast Hsp104 and its bacterial homolog, ClpB, are Clp/Hsp100 molecular chaperones and AAA+ ATPases. Hsp104 and ClpB collaborate with the Hsp70 and DnaK chaperone systems, respectively, to retrieve and reactivate stress-denatured proteins from aggregates. The action of Hsp104 and ClpB in promoting cell survival following heat stress is species-specific: Hsp104 cannot function in bacteria and ClpB cannot act in yeast. To determine the regions of Hsp104 and ClpB necessary for this specificity, we tested chimeras of Hsp104 and ClpB in vivo and in vitro. We show that the Hsp104 and ClpB middle domains dictate the species-specificity of Hsp104 and ClpB for cell survival at high temperature. In protein reactivation assays in vitro, chimeras containing the Hsp104 middle domain collaborate with Hsp70 and those with the ClpB middle domain function with DnaK. The region responsible for the specificity is within helix 2 and helix 3 of the middle domain. Additionally, several mutants containing amino acid substitutions in helix 2 of the ClpB middle domain are defective in protein disaggregation in collaboration with DnaK. In a bacterial two-hybrid assay, DnaK interacts with ClpB and with chimeras that have the ClpB middle domain, implying that species-specificity is due to an interaction between DnaK and the middle domain of ClpB. Our results suggest that the interaction between Hsp70/DnaK and helix 2 of the middle domain of Hsp104/ClpB determines the specificity required for protein disaggregation both in vivo and in vitro, as well as for cellular thermotolerance.Hsp40 | DnaJ | M-domain | GrpE | nucleotide exchange factor

Summary The Hsp90 family of heat shock proteins is an abundantly expressed and highly conserved family of ATP-dependent molecular chaperones. Hsp90 facilitates remodeling and activation of hundreds of proteins. In this study we developed a screen to identify Hsp90 defective mutants in E. coli. The mutations obtained define a region incorporating residues from the middle and C-terminal domains of E. coli Hsp90. The mutant proteins are defective in chaperone activity and client binding in vitro. We constructed homologous mutations in S. cerevisiae Hsp82 and identified several that caused defects in chaperone activity in vivo and in vitro. However, the Hsp82 mutant proteins were less severely defective in client binding to a model substrate than the corresponding E. coli mutant proteins. Our results identify a region in Hsp90 important for client binding in E. coli Hsp90 and suggest an evolutionary divergence in the mechanism of client interaction by bacterial and yeast Hsp90.

DnaK is a major heat shock protein of Escherichia coli and the homolog of hsp7O in eukaryotes. We demonstrate the mechanism by which DnaK and another heat shock protein, DnaJ, render the plasmid P1 initiator RepA 100-fold more active for binding to the P1 origin of replication.