Lon degrades stable substrates slowly but with enhanced processivity, redefining the attributes of a successful AAA+ protease

Protein quality control (pQC) in all cells is mediated by macromolecular machines that make new proteins, ensure that newly translated polypeptides are properly folded, and degrade aberrant proteins. Mechanoenzymes belonging to the AAA+ (ATPases associated with diverse cellular activities) superfamily are present in all domains of life and harness energy from chemical fuels to perform mechanical work by promoting conformational changes in other biological macromolecules. Mechanical work is required to unfold proteins and disassemble aggregates during pQC, as well as during other biological processes, such as unwinding DNA during replication and transporting cellular cargo along cytoskeletal filaments. The AAA+ protease Lon contains a AAA+ module fused to a C terminal peptidase domain and N terminal auxiliary domain, which is implicated in substrate recognition and allosteric regulation. Lon plays a major role in pQC by performing ‘housekeeping’ degradation of unfolded and misfolded proteins. Additionally, Lon recognizes and degrades natively folded proteins containing a recognition tag (degron). Detailed biochemical and biophysical characterization of Lon has lagged behind that of other AAA+ proteases, thus the underlying mechanical features of Lon catalyzed degradation were largely unknown prior to this work. In Chapter 2 of this thesis, I provide the first single¬ molecule characterization of Lon mechanical unfolding and translocation using a bacterial Lon homolog from Mycoplasma. This work reveals that Lon: (i) is a ‘powerful’ AAA+ protease given its ability to degrade very stable model substrates; (ii) has a conserved stepping mechanism shared by two related AAA+ proteases despite differences in their velocities, ATPase rates, domain architectures, and substrate contacts; and (iii) exhibits high processivity and frequently completely degrades multi domain substrates. Importantly, despite being a slower unfoldase and translocase than other well characterized AAA+ proteases, Lon is more persistent and successful at degrading targeted substrates. Based on this work, I propose that, because of its cellular niche, Lon balances degradation of a very large substrate repertoire consisting of both unfolded and highly stable proteins, and Lon has likely been ‘tuned’ during evolution to maximize its proteolytic success to a greater extent than the other bacterial AAA+ proteases. In the final chapter, I propose several future directions for the continued research on Lon both in vitro and in vivo to identify novel substrates, modes of regulation, and further elucidation of the mechanistic features of degradation.

Name thesis:degree_name

Doctoral

Department dc:contributor.department

Massachusetts Institute of Technology. Department of Biology

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2023

Author dc:creator

• Kasal, Meghann

Advisor dc:contributor.advisor

• Baker, Tania A.

Statement dc:rights

• Attribution 4.0 International (CC BY 4.0)
• Copyright retained by author(s)

Licence dc:rights.uri

• https://creativecommons.org/licenses/by/4.0/

Handle dc:identifier.uri

https://hdl.handle.net/1721.1/151628

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/151628