The destructive activities of proteases and disassembly chaperones must be controlled to ensure that they engage the proper substrates. Bacteria have no ubiquitin system, and recognition of specific protein targets for degradation or disassembly is mediated by a diverse set of unstructured peptide signals displayed on otherwise native substrate proteins. Some of these peptide signals function directly as primary degradation tags, allowing recognition and engagement by the ATPase; others function indirectly to tether the substrate to the ATPase, increasing the local concentration and the probability of engagement for degradation. The importance of “peptide” recognition was initially suggested by studies showing that mutation of a few unstructured terminal residues could dramatically change the proteolytic susceptibility of an attached protein (Bowie and Sauer, 1989
; Parsell et al., 1990
). Subsequent studies revealed that peptide signals in many substrates were responsible for their recognition by bacterial proteases or disassembly chaperones (see, for example, Keiler et al., 1996
; Laachouch et al., 1996
; Levchenko et al., 1997
; Gonciarz-Swiatek et al., 1999
; Wang et al., 1999
; Hoskins et al., 2000
; Gonzalez et al., 2000
The importance of short peptide sequences in bacterial protein destruction is exemplified by the SsrA quality control system, which adds a degradation tag to nascent proteins on stalled ribosomes (Keiler et al., 1996
). When recruited to a distressed ribosome, SsrA acts as tmRNA to direct addition of the tag sequence AANDENYALAA
to the C terminus of the nascent polypeptide. This ssrA tag, in turn, targets the attached protein for degradation by ClpXP, ClpAP, and other proteases (Keiler et al., 1996
; Gottesman et al., 1998
; Herman et al., 1998
). No additional substrate information is required for degradation, and thus the ssrA tag functions as a “strong” primary degradation signal.
In terms of protease specificity, it is natural to think about a single class of substrates being cleaved by a given enzyme, but this model is inadequate for bacterial AAA+ proteases. ClpXP, for example, degrades hundreds of different E. coli
proteins in addition to ssrA-tagged substrates, and many of these substrates interact with ClpX using different types of peptide signals (Flynn et al., 2003
). Indeed, at least five distinct classes of naturally encoded peptide motifs target proteins for degradation by ClpXP. This result can be rationalized from a biological perspective, as the existence of multiple classes of degradation signals would allow differential regulation of the degradation of disparate classes of substrates depending upon the demands of cell physiology. In fact, the intracellular levels of many ClpXP substrates change in response to oxygen levels, starvation, DNA damage, heat and cold shock, etc. (Flynn et al., 2003
). Thus, one role of AAA+ proteases may be to readjust the composition of the proteome following the global changes in gene expression that accompany responses to stress.
The existence of multiple classes of degradation signals for a single AAA+ protease raises obvious biochemical questions about how these sequences function and are recognized. Some peptide signals represent primary degradation signals, whereas others function in tethering roles. Indeed, multiple peptide signals can be present in a single substrate and be required for efficient degradation. For example, one peptide signal in the UmuD/D′ heterodimer tethers it to ClpXP, allowing efficient recognition of a second “weak” degradation signal (Neher et al., 2003b
). In a conceptual sense, the distinction between primary degradation or disassembly signals and secondary tethering signals is clear. Primary signals mediate engagement and subsequent denaturation/translocation by the AAA+ ATPase, whereas tethering signals simply increase binding affinity and the probability of engagement. Experimentally, however, determining the type of signal may not be straightforward. Eliminating either class of signal from a substrate could prevent degradation. Moreover, transplanting either type of signal to a new protein could potentially lead to its degradation. For example, fusing a tethering tag to a protein with a weak primary signal, which might comprise almost any region of unstructured polypeptide, could make the fusion protein an efficient substrate. For some substrates and AAA+ enzymes, tethering signals bind to accessory domains of the ATPase, whereas primary signals bind directly to the pore of the AAA+ ATPase (see below), but it is not yet clear whether this will be a general rule.
Degradation signals are present near the N or C terminus of a wide variety of substrates for ClpXP (Flynn et al., 2003
), and terminal recognition signals seem to be common in substrates of other proteases as well. The a-carboxylate of the ssrA tag is critical for ClpXP recognition, and signals of this type would therefore only be efficiently recognized at the C terminus of a protein (Kim et al., 2000
; Neher et al., 2003a
). Placement of signals at the beginning or end of protein sequences may also be common because these regions are often unstructured, making a degradation tag in the native protein accessible to AAA+ proteases. Some degradation tags function at either protein terminus and even at internal sites (Hoskins et al., 2002
). Hence, the key features of functional recognition signals are accessibility and the presence of apposite binding determinants.
EM (electron microscopy) studies show native substrates bound at or near the “protein-processing” pore on the protease-distal face of the ClpX ATPase (Ortega et al., 2000
). This observation plus the fact that degradation substrates must ultimately translocate through the ATPase pore raises the possibility that the pore contains the binding and engagement sites for the recognition tags of some substrates. Indeed, in some instances, ATPase pore residues have been directly implicated in substrate recognition by mutational or cross-linking studies (Song et al., 2000
; Siddiqui et al., 2004
; Schlieker et al., 2004