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Document Type

Campus-Only Access for Five (5) Years

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Molecular and Cellular Biology

Year Degree Awarded

2017

Month Degree Awarded

May

First Advisor

Peter Chien

Subject Categories

Biochemistry | Molecular Biology

Abstract

Regulated protein degradation is essential for all life. Bacteria use energy-dependent proteases to regulate protein degradation. Recognition of a substrate is enabled by the inherent specificity of the protease and by the use of adaptor proteins that widen the spectrum of recognized substrates. In Caulobacter crescentus, the timed destruction of many regulators including CtrA by the ClpXP protease drives cell cycle progression. Although, in a test tube, ClpXP can degrade CtrA by itself and does not need any helping factors, additional factors such as CpdR, RcdA and PopA are required in vivo. Understanding how these factors modulate protease activity at the mechanistic level is the major focus of this dissertation work. In this work, we show that these factors constitute an adaptor hierarchy where different substrates are destroyed based on the degree of adaptor assembly. The hierarchy builds upon priming of ClpXP by the adaptor CpdR, which promotes degradation of one class of substrates like PdeA and which also recruits the next level of adaptor RcdA to degrade a second class of substrates such as TacA. The third cyclic-di-GMP dependent adaptor PopA binds RcdA to promote destruction of a third class of substrates such as CtrA. Because adaptors must bind their cognate proteases, all adaptors run the risk of themselves being recognized as the substrate and hence degraded by the protease, a process that could limit their effectiveness. Indeed, we find that RcdA is readily degraded by CpdR-activated ClpXP protease when present alone but cargo engagement restrains its degradation. By using chimeric proteins, we find that the ability of a cargo to protect its adaptor is not due to global stabilization but is specific to the native protease recognition elements of that adaptor. We find that this principle extends across several adaptor systems, including the adaptor SspB. Together, this work reveals how hierarchical adaptors orchestrate regulated proteolysis during bacterial cell cycle progression and how, robust adaptor activity can be maintained by cargo engagement. Because of the high degree of conservation of many proteins between species, we speculate that principles found in the Caulobacter system likely generalize to others.

Available for download on Sunday, November 12, 2017

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