Abstract:
ClpXP is an Escherichia coli protease that carryout energy-dependent intracellular proteolysis. In recent years, this system has been widely studied due to its importance as a protein regulatory machinery and a virulence factor. Protein substrates of ClpXP contain degrons with a specific protein sequence. SsrA tag is one of the five degrons known to subject proteins for ClpXP degradation. SsrA is an 11 amino acid peptide added to the C-terminus of nascent polypeptide chains translated from aberrant messenger RNAs lacking stop codons via a process called trans-translation.
ClpXP was known to targets only cytosolic proteins with degrons until recently, AcrB, an E. coli membrane protein was found to be degraded by ClpXP when it is tagged by ssrA peptide, which leads to the speculation that ClpXP is capable of degrading membrane proteins. However, this speculation was challenged with the finding that ssrA tagging of ProW1−182, a different inner-membrane protein resulted in degradation by AAA+ membrane protease FtSH. We report that the membrane substrates of ClpXP bear long c-terminal cytoplasmic domains while metastable proteins lacking cytoplasmic domains are degraded by FtsH. For instance, ssrA tagged Aquaporin-Z (AqpZ), a stable tetrameric membrane protein lacking a c-terminal cytoplasmic domain is subjected to degradation by neither ClpXP nor FtsH. Nevertheless, when the c-terminus of AqpZ is fused with ssrA tagged Cyan fluorescent protein ClpXP degrades the resulted fusion protein while truncated metastable version, AqpZ 1-155 is degraded by FtSH.
This presentation also emphasizes our attempt to unravel the possible effect of proton motive force on the activity of ClpXP. We used Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) to disrupt the proton motive force. Our results suggest that degradation of soluble protein substrates such as GFP-ssrA, MurA-ssrA, Chloramphenicol acetyltransferase ssrA are not affected by CCCP. However, degradation of membrane protein substrates by ClpXP is diminished in the presence of CCCP. We speculate that either the proton motive force or ATP provided from oxidative phosphorylation is essential, or both are important for ClpXP to degrade membrane proteins.
It has been shown that the TolC is not a good target for inhibition of multidrug efflux of antibiotic-resistant bacteria as the bacterial susceptibility to antibiotics was not affected even when a significant amount of TolC is depleted. TolC is a membrane protein channel that functions in conjunction with transporters and membrane fusion proteins and provides a pathway to expel antibiotics across the E. coli outer membrane. AcrAB-TolC multidrug efflux pump is one such example where TolC cooperates with AcrB transporter and AcrA membrane fusion protein. We report that the depletion of the number of copies of AcrB makes bacteria highly susceptible to antibiotics. We utilized ClpXP degradation system to regulate the copy number of AcrB in the cell. Our results show that AcrB is an excellent target for inhibiting multidrug efflux, and ClpXP is an excellent tool to regulate antibiotic target proteins for research purposes.