Turnover of Endogenous SsrA-tagged Proteins Mediated by ATP-dependent Proteases in Escherichia coli*

Formation and degradation of SsrA-tagged proteins enable ribosome recycling and elimination of defective products of incomplete translation. We produced an antibody against the SsrA peptide and used it to measure the amounts of SsrA-tagged proteins in Escherichia coli cells without interfering with tagging or altering the context of the tag added at the ends of nascent polypeptides. SsrA-tagged proteins were present in very small amounts unless a component of the ClpXP protease was missing. From the levels of tagged proteins in cells in which degradation is essentially blocked, we calculate that ≥1 in 200 translation products receives an SsrA tag. ClpXP is responsible for ≥90% of the degradation of SsrA-tagged proteins. The degradation rate in wild type cells is ≥1.4 min–1 and decreases to ∼0.10 min–1 in a clpX mutant. The rate of degradation by ClpXP is decreased ∼3-fold in mutants lacking the adaptor SspB, whereas degradation by ClpAP is increased 3–5-fold. However, ClpAP degrades SsrA-tagged proteins slowly even in the absence of SspB, possibly because of interference from ClpA-specific substrates. Lon protease degrades SsrA-tagged proteins at a rate of ∼0.05 min–1 in the presence or absence of SspB. We conclude that ClpXP, together with SspB, is uniquely adapted for degradation of SsrA-tagged proteins and is responsible for the major part of their degradation in vivo.