Polypeptides emerging from the ribosome must fold into stable three-dimensional structures and maintain that structure throughout their functional lifetimes. Maintaining quality control over protein structure and function depends on molecular chaperones and proteases, both of which can recognize hydrophobic regions exposed on unfolded polypeptides. Molecular chaperones promote proper protein folding and prevent aggregation, and energy-dependent proteases eliminate irreversibly damaged proteins. The kinetics of partitioning between chaperones and proteases determines whether a protein will be destroyed before it folds properly. When both quality control options fail, damaged proteins accumulate as aggregates, a process associated with amyloid diseases.

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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.

The ssrA tag, an 11-aa peptide added to the C terminus of proteins stalled during translation, targets proteins for degradation by ClpXP and ClpAP. Mutational analysis of the ssrA tag reveals independent, but overlapping determinants for its interactions with ClpX, ClpA, and SspB, a specificity-enhancing factor for ClpX. ClpX interacts with residues 9–11 at the C terminus of the tag, whereas ClpA recognizes positions 8–10 in addition to residues 1–2 at the N terminus. SspB interacts with residues 1–4 and 7, N-terminal to the ClpX-binding determinants, but overlapping the ClpA determinants. As a result, SspB and ClpX work together to recognize ssrA-tagged substrates efficiently, whereas SspB inhibits recognition of these substrates by ClpA. Thus, dissection of the recognition signals within the ssrA tag provides insight into how multiple proteins function in concert to modulate proteolysis.

-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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