Proteomic Discovery of Cellular Substrates of the ClpXP Protease Reveals Five Classes of ClpX-Recognition Signals

Abstract

ClpXP is a protease involved in DNA damage repair, stationary-phase gene expression, and ssrA-mediated protein quality control. To date, however, only a handful of ClpXP substrates have been identified. Using a tagged and inactive variant of ClpP, substrates of E. coli ClpXP were trapped in vivo, purified, and identified by mass spectrometry. The more than 50 trapped proteins include transcription factors, metabolic enzymes, and proteins involved in the starvation and oxidative stress responses. Analysis of the sequences of the trapped proteins revealed five recurring motifs: two located at the C terminus of proteins, and three N-terminal motifs. Deletion analysis, fusion proteins, and point mutations established that sequences from each motif class targeted proteins for degradation by ClpXP. These results represent a description of general rules governing substrate recognition by a AAA+ family ATPase and suggest strategies for regulation of protein degradation.

A membrane metalloprotease participates in the sequential degradation of a Caulobacter polarity determinant

“…The truncated RseA fragment, RseA(DP), remains membrane-bound and continues to inhibit s E until the membrane metalloprotease YaeL makes the second cleavage; the N-terminal domain is then released into the cytoplasm for complete degradation, possibly by the ClpXP protease, relieving repression of s E (Alba et al, 2002;Kanehara et al, 2002;Flynn et al, 2003). According to our model, absence of MmpA should lead to accumulation of membrane-bound PodJ S .…”

Making the most of affinity tags

“…Sequence determinants at both the N-and C-termini of proteins can influence their rate of degradation [2][3][4]. Therefore, in some cases affinity tags might improve the yield of recombinant proteins by rendering them more resistant to intracellular proteolysis [5].…”

Unfolded protein responses in bacteria and mitochondria: A central role for the ClpXP machine

“…Consistent with a role for ClpXP in this pathway, the RsePmediated cleavage of RseA between residues 108 and 109 generates a fragment of RseA , which contains a C-motif 1 degron (VAA-COOH) for ClpX recognition (25,50). This degron targets the fragment for degradation by ClpXP, and its delivery is substantially enhanced by the action of the ClpX adaptor protein, SspB (50).…”

“…So far more than 50 candidate substrates of E. coli ClpXP have been identified including metabolic enzymes, transcription factors, and stress protective proteins (25). Some of these ClpXP substrates, that is, SsrA-tagged polypeptides, UmuD, and the general stress response transcription factor SigmaS have been examined in detail.…”