Role of communication between subunits and enzymes in ClpXP-mediated substrate unfolding and degradation

Chaperones and proteases play important roles in quality control by helping proteins fold, by dismantling hyper-stable complexes, and by degrading unwanted proteins. The highly conserved AAA+ Clp/Hsp100 proteins are ATPases which function as disassembly chaperones as well as essential components of energy-dependent proteases. For example, the ClpX ATPase disassembles macromolecular complexes and combines with the ClpP peptidase to form ClpXP, a molecular machine with structural and functional similarity to the eukaryotic 26S proteasome. ClpXP consists of hexameric ClpX rings stacked coaxially against the double-ring ClpP₁₄ peptidase. ClpXP's peptidase active sites reside in a sequestered chamber accessible through a narrow channel which excludes native, folded substrates. ClpX binds specific substrates, unfolds them in a reaction requiring ATP hydrolysis, and then translocates them into ClpP for degradation. When ClpP is absent, ClpX unfolds and releases specific substrates, an activity that can disassemble otherwise stable complexes. Although ClpX-mediated substrate unfolding and ClpXP-mediated degradation have been studied extensively, the role of communication between subunits within a ClpX hexamer or between enzymes in ClpXP has not been addressed. I initially screened numerous ClpX point mutants for their ability to support host-cell lysis by bacteriophage Mu, and then purified several mutants for functional characterization in vitro. Three of these mutants contained substitutions at intersubunit interfaces; these variants bound substrate well, but displayed unusual changes in ATP hydrolysis in response to substrate binding and had low protein unfolding activity.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Biology.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2004

Author dc:creator

• Joshi, Shilpa Arun, 1975-

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/1721.1/16608

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/16608