Date of Award


Degree Type


Degree Name

Doctor of Philosophy in Biological and Environmental Sciences


Cell and Molecular Biology (CMB)


Cell & Molecular Biology

First Advisor

Jodi L. Camberg


The AAA+ (ATPases associated with a variety of cellular activities) protein superfamily includes approximately 30,000 molecular machines powered by adenosine triphosphate (ATP) binding and hydrolysis events, which are important for many cellular processes, including cell division, DNA replication, intracellular transport, and protein quality control. The bacterial AAA+ protein ClpXP is a two component ATP-dependent chaperone-protease that recognizes protein substrates bearing specific recognition signals, subsequently unfolding and degrading them to eliminate unnecessary or misfolded proteins. Since degradation is irreversible, highly specific recognition motifs are needed to ensure intentional engagement. A common strategy to facilitate recognition of substrates by AAA+ ATPases is to display multivalent recognition motifs, usually as a result of oligomerization or polymerization. During cell division in Escherichia coli, it has been reported that ClpXP degrades the essential cell division protein FtsZ (the prokaryotic tubulin homolog) in both the monomer and polymer form, however the degrons important for ClpX recognition of FtsZ, the mechanism by which ClpX recognizes and degrades FtsZ, and the physiological relevance for this regulated proteolytic event were previously unknown. We review more about substrate discrimination, multivalent recognition, and processive unfolding of FtsZ by ClpXP in Manuscript I.

In Manuscript II, we identified regions important for ClpXP targeting the native substrate FtsZ. First, we mutagenized FtsZ mutant proteins and degraded these proteins in vitro to deduce the regions (known as degrons) important for degradation by ClpXP. Then, we examined relevant Gfp-tagged FtsZ mutant proteins in dividing E. coli cells in vivo to further understand the importance of impairing regulated proteolysis on FtsZ assembly and cell division.

In Manuscript III, we propose the mechanism for ClpX recognition of FtsZ. We performed a traditional degradation assay with custom protein substrates in vitro to deduce the requirements for each degron for ClpX recognition of a monomer or polymer of FtsZ. Taken together, Manuscripts II and III describe a differential, dual-targeting role for a AAA+ substrate that will provide mechanistic insight in the field for degradation strategies in the crowded, cellular milieu.

ClpXP degrades approximately 15% of total FtsZ per cell cycle, and therefore regulated proteolysis is the proposed role of ClpXP in cell division, however, ClpXP is not essential for this process. In Manuscript IV, we examined the physiological relevance of ClpXP proteolysis during cell division in E. coli by performing photobleaching and recovery assays on Gfp-tagged FtsZ structures and measured the half-time recovery in clp-deficient strains or wildtype cells containing Gfp-tagged FtsZ mutant proteins used for Manuscript II. For the first time, we established a phenotype for cell division when regulated proteolysis was impaired using these approaches and implicated the recognition region of FtsZ by ClpX, which is shared by other modulatory cell division proteins, in the importance of septation.

Finally, using our understanding from the work described for Manuscript II, we demonstrated in Manuscript V that ClpXP recognizes and degrades aggregated FtsZ in vitro and requires the known degrons for degradation in the aggregated state. We show that recognizing FtsZ aggregates in vivo is important since there are higher FtsZ levels in clp-deficient cells compared to wild type after heat shock. Furthermore, ClpXP recognizes and degrades the engineered Gfp-ssrA substrate when aggregated, and that the chaperone ClpX alone promotes the reactivation of aggregated Gfp-ssrA. In conclusion, we describe a novel role for ClpXP under stressful cellular conditions and ClpX alone in disaggregation for substrates in E. coli, which broadens our understanding of the role of ClpXP in proteostasis.



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