Tuning strength and specificity in the N-end rule

Protein degradation is a central mechanism in the regulation of gene expression and activity. Proteolysis regulates not only homeostatic activities, but also the cell's responses to stress. A recurring question underlying this regulatory process is the specificity of substrate selection by the proteolytic machinery. I designed an unbiased selection to isolate N-terminal degradation sequences in vivo, which led to a collection of N-end rule signals. The N-end rule describes how the identity of a protein's N-terminal residue determines its metabolic stability. In E. coli, CIpAP is the principal protease that degrades proteins bearing an N-terminal phenylalanine, tyrosine, tryptophan, or leucine residue. The CIpS adaptor, which displays homology to eukaryotic ubiquitin ligases that recognize N-end signals, is a recently discovered component of the bacterial N-end rule. Using the collection of N-end signals, I was able to demonstrate that ClpS enhances N-end degradation by ClpAP but is not required in vivo or in vitro. The collection of N-end signals also provided insight into the role of sequence context in the N-end rule. Specifically, acidic residues and the length of the N-end signal affect degradation rates in vitro. These defective N-end signals also allowed us to separately define recognition specificities of ClpS and ClpAP. Whereas ClpS bound poorly to acidic N-end signals, CIpAP was unable to degrade substrates with short N-end sequences. Although two decades of biochemical and cellular data support the importance of the Nterminal residue in N-end degradation, there has been no structural information explaining how a single residue is recognized as a degradation signal.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Biology.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2008

Author dc:creator

• Wang, Kevin H., Ph. D. Massachusetts Institute of Technology

Advisor dc:contributor.advisor

• Tania A. Baker.

• 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/1721.1/45808

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/45808