| dc.identifier.uri | http://hdl.handle.net/11401/78276 | |
| dc.description.sponsorship | This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree. | en_US |
| dc.format | Monograph | |
| dc.format.medium | Electronic Resource | en_US |
| dc.language.iso | en_US | |
| dc.type | Dissertation | |
| dcterms.abstract | The i-AAA protease is a membrane-anchored ATP-dependent protease that contributes to proteostasis of the mitochondrial inner membrane and intermembrane space. The protease is required to select specific substrates for degradation from among the diverse complement of proteins present in mitochondria, yet the molecular mechanisms of substrate recognition have yet to be fully described. Importantly, dysfunction of the mitochondrial AAA proteases has been implicated in the development of numerous neurodegenerative diseases. Detailed solution studies of these enzymes have been hampered by the requirement for an insoluble transmembrane span to drive hexamerization and thus form the ATPase active sites. By replacing the transmembrane domain with a soluble hexameric coiled-coil, we have developed a method for assembling soluble, active, hexameric human and yeast i-AAA proteases, YME1L and Yme1p, respectively, that are fully competent for ATP-dependent protein degradation in vitro. Utilizing these rebuilt proteolytic systems I have determined general principles of substrate recognition and processing for the i-AAA protease. In addition to requiring an accessible recognition sequence, the i-AAA protease is able to discriminate between degradation signals by their amino acid composition, implying the use of sequence-specific signals in mitochondrial proteostasis. Furthermore, the protease is able to processively unfold and degrade substrates with varying thermodynamic stabilities. These findings are substantiated by the identification of a novel degron sequence that targets the Tim10 subunit of the Tim9/10 mitochondrial translocase complex for proteolysis by i-AAA. Intriguingly, this degron is necessary and sufficient to direct model proteins for degradation by i-AAA and represents the first confirmed degradation sequence for substrates of the mitochondrial AAA proteases. Moreover, I show how reductive loss of conserved disulfide bonds within Tim10 accelerates its degradation by i-AAA. Together, these results significantly advance our understanding of substrate recognition by the mitochondrial AAA proteases and suggest a mechanism for proteolytic control of mitochondrial proteins of the intermembrane space. | |
| dcterms.available | 2018-06-21T13:38:51Z | |
| dcterms.contributor | Glynn, Steven E | en_US |
| dcterms.contributor | Bogenhagen, Daniel | en_US |
| dcterms.contributor | Karzai, Wali | en_US |
| dcterms.contributor | Luk, Ed | en_US |
| dcterms.contributor | Wiseman, Luke | en_US |
| dcterms.creator | Rampello, Anthony John | |
| dcterms.dateAccepted | 2018-06-21T13:38:51Z | |
| dcterms.dateSubmitted | 2018-06-21T13:38:51Z | |
| dcterms.description | Department of Molecular and Cellular Biology | en_US |
| dcterms.extent | 105 pg. | en_US |
| dcterms.format | Monograph | |
| dcterms.format | Application/PDF | en_US |
| dcterms.identifier | http://hdl.handle.net/11401/78276 | |
| dcterms.issued | 2017-12-01 | |
| dcterms.language | en_US | |
| dcterms.provenance | Made available in DSpace on 2018-06-21T13:38:51Z (GMT). No. of bitstreams: 1
Rampello_grad.sunysb_0771E_13578.pdf: 26268234 bytes, checksum: eb53c4cdb3d983c8e7d4fb363a36f023 (MD5)
Previous issue date: 12 | en |
| dcterms.subject | AAA + | |
| dcterms.subject | Biochemistry | |
| dcterms.subject | degron | |
| dcterms.subject | Biology | |
| dcterms.subject | intermembrane space | |
| dcterms.subject | mitochondria | |
| dcterms.subject | protein quality control | |
| dcterms.title | Defining the rules of substrate recognition for a mitochondrial AAA+ protease | |
| dcterms.type | Dissertation | |
