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

Tawfilis, Sherif. Identification and analysis of Clp protease substrates in "C. crescentus". 2004, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_6924

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Abstract

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.
Advisors:Jenal, Urs
Committee Members:Bickle, Thomas A. and Dehio, Christoph
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Infection Biology > Molecular Microbiology (Jenal)
05 Faculty of Science > Departement Biozentrum > Growth & Development > Molecular Microbiology (Jenal)
UniBasel Contributors:Jenal, Urs and Dehio, Christoph
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:6924
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:157
Language:English
Identification Number:
Last Modified:22 Jan 2018 15:50
Deposited On:13 Feb 2009 14:56

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