ClpXP, a bacterial AAA+ protease, controls intracellular levels of many stress-response proteins. To investigate substrate profile changes caused by a specific environmental stress, quantitative mass spectrometry (SILAC) was used to analyze proteins trapped by ClpXP(trap) before and after DNA damage. The abundance of half of the trapped proteins changed more than 3-fold after damage. Overrepresented substrates included the DNA-repair proteins RecN and UvrA. Among SOS-response proteins, 25% were ClpXP substrates and, importantly, nearly all of the highly induced regulon members were rapidly degraded. Other proteins, including the stress regulator sigma(S), were underrepresented in ClpXP(trap) after DNA damage; overproduction experiments suggest that simple substrate competition does not account for this reduced recognition. We conclude that damage-response proteins are an unusually rapidly degraded family and that ClpXP has substantial capacity to process the influx of newly synthesized substrates while maintaining the ability to degrade its other substrates in an environmentally responsive manner.

Clp/Hsp100 ATPases remodel and disassemble multiprotein complexes, yet little is known about how they preferentially recognize these complexes rather than their constituent subunits. We explore how substrate multimerization modulates recognition by the ClpX unfoldase using a natural substrate, MuA transposase. MuA is initially monomeric but forms a stable tetramer when bound to transposon DNA. Destabilizing this tetramer by ClpX promotes an essential transition in the phage Mu recombination pathway. We show that ClpX interacts more tightly with tetrameric than with monomeric MuA. Residues exposed only in the MuA tetramer are important for enhanced recognition--which requires the N domain of ClpX--as well as for a high maximal disassembly rate. We conclude that an extended set of potential enzyme contacts are exposed upon assembly of the tetramer and function as internal guides to recruit ClpX, thereby ensuring that the tetrameric complex is a high-priority substrate.

Sequences are reported for portions of two mitochondrial genes from a domestic horse and a plains zebra and compared to those published for a quagga and a mountain zebra. The extinct quagga and plains zebra sequences are identical at all silent sites, whereas the horse sequence differs from both of them by 11 silent substitutions. Postmortem changes in quagga DNA may account for the two coding substitutions between the quagga and plains zebra sequences. The hypothesis that the closest relative of the quagga is the domestic horse receives no support from these data. From the extent of sequence divergence between horse and zebra mitochondrial DNAs (mtDNAs), as well as from information about the fossil record, we estimate that the mean rate of mtDNA divergence in Equus is similar to that in other mammals, i.e., roughly 2% per million years.