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Turnover of endogenous SsrA-tagged proteins mediated by ATP-dependent proteases in Escherichia coli.

ABSTRACT: 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 > or =1 in 200 translation products receives an SsrA tag. ClpXP is responsible for > or =90% of the degradation of SsrA-tagged proteins. The degradation rate in wild type cells is > or =1.4 min(-1) and decreases to approximately 0.10 min(-1) in a clpX mutant. The rate of degradation by ClpXP is decreased approximately 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 approximately 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.

SUBMITTER: Lies M

PROVIDER: S-EPMC2516991 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundOverexpression of recombinant proteins usually triggers the induction of heat shock proteins that regulate aggregation and solubility of the overexpressed protein. The two-dimensional gel electrophoresis (2-DE)-mass spectrometry approach was used to profile the proteome of Escherichia coli overexpressing N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase), both fused to glutathione S-transferase (GST) and polyionic peptide (5D or 5R).ResultsOverexpression of fusion proteins by IPTG induction caused significant differential expression of numerous cellular proteins; most of these proteins were down-regulated, including enzymes connected to the pentose phosphate pathway and the enzyme LuxS that could lead to an inhibition of tRNA synthesis. Interestingly, when plasmid-harboring cells were cultured in LB medium, gluconeogenesis occurred mainly through MaeB, while in the host strain, gluconeogenesis occurred by a different pathway (by Mdh and PckA). Significant up-regulation of the chaperones ClpB, HslU and GroEL and high-level expression of two protective small heat shock proteins (IbpA and IbpB) were found in cells overexpressing GST-GlcNAc 2-epimerase-5D but not in GST-Neu5Ac aldolase-5R-expressing E. coli. Although most of the recombinant protein was present in insoluble aggregates, the soluble fraction of GST-GlcNAc 2-epimerase-5D was higher than that of GST-Neu5Ac aldolase-5R. Also, in cells overexpressing recombinant GST-GlcNAc 2-epimerase-5D, the expression of σ32 was maintained at a higher level following induction.ConclusionsDifferential expression of metabolically functional proteins, especially those in the gluconeogenesis pathway, was found between host and recombinant cells. Also, the expression patterns of chaperones/heat shock proteins differed among the plasmid-harboring bacteria in response to overproduction of recombinant proteins. In conclusion, the solubility of overexpressed recombinant proteins could be enhanced by maintaining the expression of σ32, a bacterial heat shock transcription factor, at higher levels during overproduction.

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Turnover of endogenous SsrA-tagged proteins mediated by ATP-dependent proteases in Escherichia coli.

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