Specificity and regulation of substrate degradation for a AAA+ protease

Energy dependent proteolysis is a critical method of cellular regulation for all forms of life. The AAA+ proteases ClpXP and ClpAP in E. coli function in this capacity by facilitating the denaturation and degradation of target substrates. These proteolytic enzymes degrade hundreds of different proteins. Determining how the activities of these proteases are regulated in the cell as well as learning how these enzymes bind and engage substrates are important goals. In order to better understand how the degradation of ClpXP and ClpAP is regulated, I studied their contributions to ssrA-tagged protein degradation in the cell. Using GFP-ssrA expressed from the chromosome as a degradation reporter, the effects of altered concentrations of different protease components or adaptor proteins were explored. I found that both ClpXP and ClpAP could degrade GFP-ssrA in the cell and that increased levels of ClpAP in stationary phase resulted in increased degradation of ssrA-tagged substrates. I also demonstrated that wild-type levels of the adaptor proteins SspB and ClpS do not fully inhibit ClpAP degradation of GFP-ssrA. To better understand how the ClpXP enzyme binds substrates, I took a mutagenic approach.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Biology.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2007

Author dc:creator

• Farrell, Christopher Mark

Advisor dc:contributor.advisor

• Robert T. Sauer.

• Biology.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/38998

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/38998