Noncanonical recognition and degradation of a stable soluble protein by AAA protease FtsH

Abstract

AAA+ (ATPases associated with various cellular activities) proteolytic machines help maintain and adjust the cellular proteome in response to stress or changes in nutrients. AAA+ proteases bind degradation targets and utilize ATP-powered conformational changes in the AAA+ unfoldase ring to denature and translocate the substrate polypeptide into an associated, sequestered protease chamber for degradation. Of the five AAA+ proteases in Escherichia coli, FtsH is unique in being genetically essential, in its localization to the membrane, and in its function in degrading both membrane and cytosolic proteins. Prior in vitro characterization suggested that FtsH only degrades meta-stable proteins despite its ability to extract protein substrates from the membrane for degradation. These results motivated me to reinvestigate the determinants in a substrate required for effective FtsH unfolding. In this thesis, I present experiments that first test the hypothesis that FtsH may unfold and degrade a more stable protein in vitro with a sufficiently long degradation tag (degron) and then explore noncanonical recognition as one mechanism that may be employed in FtsH-dependent degradation.

In Chapter I, I review our current understanding of AAA+ protease structure and function, especially as it pertains to FtsH to provide background for the later chapters. In Chapter II, I test the hypothesis that a long degron may be required for FtsH to successfully bind and unfold E. coli dihydrofolate reductase (DHFR), a stable protein which was previously found to resist FtsH degradation. Strikingly, I find that detergent-solubilized FtsH can degrade DHFR in vitro with or without an appended degron. I then show that FtsH recognition of DHFR is noncanonical and not dependent on unstructured terminal degrons but suggest a model for how FtsH may unfold DHFR by engaging an internal site in a partially unfolded intermediate. In Chapter III, I test the hypothesis that FtsH may bind another stably folded soluble protein, cyclopropane fatty acid synthase (CFAS), at an internal site. In Chapter IV, I propose future directions that may further enrich our understanding of FtsH-DHFR degradation and experimental approaches that can be applied to assess the kinetics of assembly/disassembly of other enzyme-substrate complexes.