Identification and analysis of Clp protease substrates in "C. crescentus"

Proteolysis is an irreversible regulatory mechanism by which cells can remove a protein whose function is no longer required or if its presence in a particular cellular compartment and/or at a certain time is harmful to the cell. Degradation of cytoplasmic proteins is energy dependent, and in prokaryotic cells is carried out by five ATP-dependent proteases, namely, ClpXP, ClpAP, FtsH, Lon and HslUV (Gottesman and Maurizi 1992). Protein degradation has been shown to be of crucial importance for a variety of cellular processes such as stress response, DNA damage repair and cell cycle progression (Jenal and Fuchs 1998; Jenal and Hengge-Aronis 2003; Straus et al. 1988; Sutton et al. 2000). A critical event occurring during the cell cycle progression of C. crescentus is the degradation of the essential master cell cycle regulator, CtrA, during the G1-to-S phase transition (Quon et al. 1996). CtrA belongs to the response regulator superfamily of proteins and aside from directly controlling the expression of about 100 genes (Laub et al. 2002; Laub et al. 2000) it suppresses DNA replication initiation by binding to several sites in the origin of replication (Quon et al. 1998). CtrA is degraded in a ClpXP-dependent manner as depletion of ClpXP results in its stabilisation. Furthermore, in the absence of ClpXP, cells are arrested at the G1 phase of the cell cycle, become filamentous and lose viability (Jenal and Fuchs 1998). A similar phenotype is observed in cells expressing a stable and constitutively active variant of CtrA (Domian et al. 1997). Inhibition of CtrA degradation alone does not cause a G1 cell cycle block, suggesting that the G1 arrest observed in cells depleted for ClpXP is not due to CtrA stabilisation. This suggests that the ClpXP-dependent degradation of one or several additional proteins is essential for cell cycle progression and survival. The primary aim of this work was to identify novel substrates of the ClpXP protease, particularly those whose timed degradation is critical for G1-to-S phase transition. This task is of crucial importance as previous work by Grünenfelder et al. (2001) has shown that a large fraction of the cell’s proteins is rapidly degraded and differentially synthesised during the cell cycle. It is likely that a subset of these proteins is involved in cell cycle progression and control. In the first part of this thesis (Chapter 3) we created a vector that allowed the conditional expression of the dominant negative allele of clpX, clpXATP, for use in a global proteomics screen to identify ClpXP substrates. In this screen, proteins that became stable upon disruption of ClpX activity were to be identified. The rationale behind our generating the conditional clpXATP allele was to create a system wherein ClpX activity can be rapidly disrupted in cells. The original ClpX depletion strain created by Jenal and Fuchs (1998) required at least four hours before the ClpX protein is undetectable in cells. Since the result of ClpX depletion is ultimately cell death, use of this mutant in a proteomics screen would make it difficult to distinguish between proteins stabilised as a direct effect of ClpX depletion and those stabilised as a consequence of cell deterioration. We found that expression of clpXATP, which has mutations in the walker A motif of the ATPase domain, results in rapid CtrA stabilisation, cell elongation and cell death. We propose that this is due to ClpXATP monomers inactivating ClpX through the formation of mixed oligomers with ClpXwt monomers. As this study was in progress, the crystal structure of ClpX from H. pylori was solved revealing that the residues we mutated in ClpXATP contact the ATP moiety and may be involved in ATP hydrolysis (Kim and Kim 2003). Thus, our results indicate that the presence of ClpXATP disrupts ClpX activity by preventing the assembly of hexameric rings, disturbing ATP binding and/or inhibiting ATP hydrolysis by the mixed ClpX hexamers. Although the nature and oligomeric state of the mixed oligomers is not clear from our results, previous work with clpA alleles with similar mutations in the walker A motif has demonstrated that mixed hexamers do form and that monomer swapping readily occurs (Seol et al. 1995; Singh and Maurizi 1994). Through the global comparison of protein stability between wild-type and clpXATP expressing cells, we found nine proteins to be stabilised as a result of ClpX inactivation. These include CtrA and CheD, both previously identified as ClpXP substrates using genetic means (Jenal and Fuchs 1998; M.R.K. Alley, unpublished). Target validation confirmed that CtrA, CheD and the product of the CC2323 gene were degraded in a ClpXP-dependent manner. CC2323 is a protein of unknown function whose orthologues are found exclusively in alpha proteobacteria. CC2323 expression was previously found to be regulated by GcrA, a cell cycle regulator that inversely oscillates with CtrA (Holtzendorff et al. 2004). We found that CC2323 synthesis is limited to the late S-, and G2- phase of the cell cycle and that its product is rapidly degraded. As a result, the CC2323 protein is only present when it is actively synthesised and is therefore absent in SW and ST cells. Our results indicate that CC2323 may be degraded by ClpXP and that its levels during the cell cycle are controlled only through its regulated expression. Although CC2323 was found to be non-essential for growth, our results indicate that its overproduction is deleterious for cell growth and survival. Thus, it appears that either high levels of CC2323, or its undesirable presence in certain cellular compartments and/or phases of the cell cycle, have negative effects on the cells. Future analysis will aim to address the reasons why CC2323 overproduction is harmful to cells and why its cellular concentration appears stringently controlled during the cell cycle at the levels of both expression and proteolysis. In the second part of this thesis (Chapter 4) we defined SsrA-tagged proteins as additional targets of the ClpXP protease in C. crescentus, and conducted a functional examination of the SsrA tag. The SsrA is a protein tag that is attached to proteins under a variety of conditions, including starvation, and targets them for degradation by ATP-dependent proteases. In E. coli, ClpXP is the main protease that is responsible for SsrA-tagged substrate degradation (Gottesman et al. 1998). We constructed several fusions between FlbD, a transcriptional regulator of late flagellar genes, and the SsrA to determine if in C. crescentus, as in E. coli, ClpXP degrades SsrA tagged substrates. FlbD-SsrA was found to be highly unstable but was stabilised upon induction of the clpXATP allele. Similarly, FlbD-SsrA was stabilised when ClpP was depleted from cells. This indicated that ClpXP is responsible for the rapid turnover of SsrA-tagged proteins in C. crescentus. SsrA-tagged FlbD variants were then used to genetically dissect the SsrA degradation pathway. We found that cells bearing FlbD-SsrA were non-motile due to the rapid degradation of FlbD and consequent lack of flagellar gene expression. To identify mutations, cis or trans, that stabilised FlbD-SsrA, a selection for motile suppressors was carried out. Our hypothesis was that cells which regained motility would have stabilised FlbD through mutations in the SsrA tag or in an accessory component. Only two suppressors were isolated that contained amino acid substitutions in the SsrA tag, indicating that these are important residues for recognition by ClpX. The remainder of the motile suppressors contained deletion or insertion frame-shifts by which the identity of the FlbD C-terminus was completely altered and the SsrA tag removed. In most cases, this resulted in FlbD stabilisation. However, transfer of one of those alleles into a clean genetic background suggested that the flbD allele alone is not able to restore motility. From this we concluded that FlbD variants with an altered C-terminus were non-functional and that a second mutation in trans must have occurred to restore motility. Consistent with this, FlbD fused to a stable variant of SsrA (FlbD-SsrADDD) did not support motility. Motile suppressors of strains carrying FlbD-SsrADDD had retained the nature of their SsrA tag, again suggesting that mutations in trans had restored motility. Those could map to components that either regulate the activity of FlbD or interact with it. It will be interesting to map these mutations as they may provide useful information about FlbD and its regulation of flagellar assembly in C. crescentus. The challenge for future work will be to map the second site mutation(s) and to define the exact contributions of cis- and transmutations for FlbD stability and/or activity. In the third and final part of this thesis (Grünenfelder et al. 2004), we examined cell cycle-dependent FliF degradation. FliF forms the MS ring that anchors the flagellum in the inner membrane. Degradation of FliF at the G1-to-S phase transition coincides with flagellar ejection and was hypothesised to be the committing step of this developmental process (Grünenfelder et al. 2003; Jenal and Shapiro 1996). We found that the non-essential ClpAP protease is required for the degradation of FliF as SW cells differentiate into ST cells. To define the nature of the ClpAP degradation signal, we conducted a high resolution mutational analysis of the FliF C-terminus. We found that though the degradation signal of FliF resides in the last 28 residues of the protein, no primary sequence appears to govern its turnover. Instead, our results indicate a requirement for hydrophobic residues at the C-terminus of FliF.