Mechanism of active substrate delivery by the AAA+ protease adaptor CIpS

AAA+ molecular machines power myriad cellular processes including protein degradation, microtubule severing, membrane fusion, and initiation of DNA replication. Protein quality control in all organisms involves deployment of ATP-dependent proteases, consisting of hexameric AAA+ rings that unfold and translocate specific substrates into an associated peptidase barrel. Adaptor proteins assist in recognition and degradation of certain substrates, but how enzyme-adaptor pairs ensure proper substrate selection is incompletely understood. In this thesis I focus on the delivery mechanism employed by the bacterial adaptor protein CIpS. The CIpS adaptor collaborates with the AAA+ CIpAP protease to recognize and degrade N-end rule substrates. CIpS binds the substrate's N-degron and assembles into a high-affinity CIpS-substrate-CIpA complex, but how the N-degron is transferred from CIpS to the axial pore of the AAA+ CIpA unfoldase to initiate degradation is not known. Here, we demonstrate that the unstructured N-terminal extension (NTE) of CIpS enters the CIpA processing pore in the active ternary complex and that CIpA engagement of the CIpS NTE is crucial for CIpS-mediated substrate delivery. In addition, I report evidence that CIpA engagement of the CIpS NTE drives structural rearrangements in CIpS important for N-end rule substrate delivery. Furthermore, our preliminary experiments suggest that CIpS is able to resist degradation by CIpAP due to a combination of a high local stability and a challenging translocation sequence at the junction of the NTE and folded core domain. I propose a model in which CIpA remodels CIpS by translocating the NTE, triggering delivery of the N-end rule substrate. Similar mechanisms may be employed by other AAA+ enzymes that collaborate with adaptor proteins to remodel/disassemble substrates without destroying them by degradation.

Department dc:contributor.department

Massachusetts Institute of Technology. Department of Biology.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2015

Author dc:creator

• Rivera-Rivera, Izarys

Advisor dc:contributor.advisor

• Tania A. Baker and 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/1721.1/101352

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/101352