Function and Control of the Spx-Family of Proteins Within the Bacterial Stress Response by Peter Zuber

Author:Peter Zuber
Language: eng
Format: epub
Publisher: Springer New York, New York, NY

Post-Transcriptional Control of Spx: Regulated Proteolysis

Western blot analysis of Spx after thiol stress induced by diamide treatment of B. subtilis cells resulted in a transient increase of Spx protein concentration, with protein levels increasing after 10 min and subsiding after 60 min post-treatment (Nakano et al. 2003a). The same pattern was observed when the spx gene was expressed from an IPTG-inducible promoter, indicating that the control of Spx concentration was exerted at the post-transcriptional level. Accumulating evidence pointed to the ATP-dependent protease, ClpXP, as an important player in post-transcriptionally controlling Spx concentration (Nakano et al. 2001, 2002, 2003b). This was supported by the aforementioned phenotype of the clpX mutant and the in vitro reconstruction of Spx proteolysis catalyzed by ClpXP. Rapid degradation of Spx by ClpCP was demonstrated in vitro when one of the adaptor proteins, MecA or YpbH, was present (Nakano et al. 2002). Mutations in clpC or mecA had little effect on the level of Spx in vivo, however, suggesting that ClpXP proteolysis is masking the Spx-targeted activity of ClpCP. That SpxA is barely detectable in cells during unperturbed growth in culture is because ClpXP catalyzes its degradation.

As with other ClpXP substrates, the C-terminal end of B. subtilis SpxA was observed to be necessary for Spx to serve as a substrate. When the C-terminal end of Spx was changed from LAN to LDD, Spx was protease resistant and could be produced in high concentration in B. subtilis cells (Nakano et al. 2003b). Both SpxA1 and SpxA2 of B. anthracis could also be produced to high concentration by replacing the AN residues at the C-terminus with DD (S. Barendt, M. M. Nakano, and P. Zuber, unpublished). MgsR appears to be a substrate for ClpXP proteolysis based on the B. subtilis clpX mutant phenotype, but does not share C-terminal residue sequence homology with Spx (Reder et al. 2012d). The C-terminal end of SpxA is highly conserved among orthologs of Bacillales, but the Staphylococcus sp. bear a VD at the end instead of AN, and the L at the third position from the end is often replaced with M. It is not known how these changes might affect proteolytic control of Spx if it operates in other members of Bacillales.

Adaptor proteins are substrate recognition factors that bind substrate proteins and tether them to the protease, thus accelerating the rate of proteolysis (Kirstein et al. 2009). For example, MecA and YpbH function as adaptors, or substrate recognition factors, for ClpCP-catalyzed proteolysis in B. subtilis (Persuh et al. 2002; Schlothauer et al. 2003). The most studied adaptor of ClpXP is the SspB protein of E. coli, which recognizes the SsrA tag appended to products of interrupted translation by tmRNA (Hersch et al. 2004). SspB makes contact with both substrate and ClpX to facilitate delivery of the substrate for proteolysis. The discovery of a mutation in the yjbH gene that results in elevated SpxA concentration without affecting spx gene transcription (Larsson et al. 2007) suggested that the yjbH product might serve an adaptor/chaperone function.

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