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Biochemical and Genetic Studies of the N-End Rule Pathway in Yeast and Mammals

Citation

Wadas, Brandon Christopher (2016) Biochemical and Genetic Studies of the N-End Rule Pathway in Yeast and Mammals. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Z91V5BZQ. http://resolver.caltech.edu/CaltechTHESIS:06022016-162251299

Abstract

Regulation of the in vivo half-lives of intracellular proteins is an important cellular process. Many intracellular proteins are short-lived, owing to their regulated and processive degradation by the Ubiquitin (Ub)-Proteasome System (UPS). In eukaryotes, the N-end rule pathway is one specific pathway within the UPS. The N-end rule pathway relates the identity of the N-terminal residue of a protein, or a protein fragment, to its in vivo half-life. Substrates of the N-end rule pathway are recognized by the presence of degradation signals, termed N-degrons. Recognition components of the N-end rule pathway are E3 ubiquitin ligases that are capable of binding to N-degrons. The N-end rule pathway consists of two distinct branches: the Arg/N-end rule pathway and the Ac/N-end rule pathway.

In the present studies, we demonstrate a complementary targeting of the rat serotonin N-acetyltransferase (AANAT), an important mediator of circadian physiology, by both branches of the N-end rule pathway. The co-targeting results from incomplete N-terminal (Nt-) acetylation of a Met-Ф motif at the N-terminus of AANAT in vivo. In the same study, we demonstrate that human AANAT is substantially longer-lived than its rat counterpart, owing to differences in their N-terminal sequences. This molecular genetic investigation of the degradation of a physiological N-end rule substrate followed an analogous earlier study, in which we reported that a clinically-relevant (blood pressure-increasing) Q2L mutant of human RGS2 (termed ML-RGS2), a regulator of G proteins, could likewise be co-targeted by both branches of the N-end rule pathway. Together, AANAT and RGS2 are the first identified and characterized physiological substrates of the Ac/N-end rule pathway in mammals.

We also report on the development and use of in vitro N-terminal arginylation (Nt-arginylation) assays using CelluSpots peptide arrays, in conjunction with pulse-chase assays in rabbit reticulocyte extract, for the systematic investigation of the effects of N-terminus-proximal sequence context on the Nt-arginylation activity of the Ate1 arginyltransferase, a component of the Arg/N-end rule pathway. These experiments help to define the sequence requirements for efficient Nt-arginylation by Ate1. Finally, we demonstrate that Rec8, a subunit of the cohesin protein complex during meiosis, is a natural short-lived substrate of the mammalian Arg/N-end rule pathway.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:AANAT; ubiquitin; ubiquitylation; N-terminal arginylation; protein degradation; Ubr1; N-End Rule Pathway
Degree Grantor:California Institute of Technology
Division:Biology and Biological Engineering
Major Option:Biology
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Varshavsky, Alexander J.
Thesis Committee:
  • Deshaies, Raymond Joseph (chair)
  • Dunphy, William G.
  • Shan, Shu-ou
  • Varshavsky, Alexander J.
Defense Date:31 May 2016
Record Number:CaltechTHESIS:06022016-162251299
Persistent URL:http://resolver.caltech.edu/CaltechTHESIS:06022016-162251299
DOI:10.7907/Z91V5BZQ
Related URLs:
URLURL TypeDescription
http://10.1126/science.aaa3844DOIArticle adapted for Ch. 3
http://10.1074/jbc.M116.714964DOIArticle adapted for Ch. 5
http://10.1073/pnas.1419587111DOIArticle adapted for Ch. 6
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:9826
Collection:CaltechTHESIS
Deposited By: Brandon Wadas
Deposited On:06 Jun 2016 20:23
Last Modified:18 May 2017 21:53

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