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A CDC48 COFACTOR AFFECTS UBIQUITINATED PROTEIN LEVELS, ENDOPLASMIC RETICULUM ASSOCIATED DEGRADATION AND PROTEASOME SUBTYPES

Tran, Joseph R (2013) A CDC48 COFACTOR AFFECTS UBIQUITINATED PROTEIN LEVELS, ENDOPLASMIC RETICULUM ASSOCIATED DEGRADATION AND PROTEASOME SUBTYPES. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

The ubiquitin proteasome system (UPS) maintains cellular homeostasis by controlling the turnover of important regulatory enzymes and by the removal of damaged or misfolded proteins. The UPS serves as the basic framework of many specific catabolic pathways, including the Endoplasmic Reticulum-Associated Degradation (ERAD) pathway, which in particular helps to maintain the homeostasis of the early secretory pathway. An important component of the ERAD pathway is a complex containing the AAA ATPase, Cdc48p. The Cdc48p complex couples ATP hydrolysis with the physical dislocation of ubiquitinated substrates from the endoplasmic reticulum prior to degradation by a multi-subunit catabolic protease known as the 26S proteasome. Here I show that the loss of a Cdc48p cofactor, VCP/Cdc48p-associated Mitochondrial Stress-responsive-1 (Vms1p), negatively affects the turnover of the model ERAD substrate, the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), but does not affect the ubiquitination of CFTR. Strains lacking both the VMS1 gene and other select Cdc48p cofactors, namely genes encoding members of the Ubiquitin Regulatory X and Ubiquitin Fusion Degradation families, are hypersensitive to certain chemical stressors, and also display additive ERAD defects for certain substrates. Curiously, VMS1 mutants show increased accumulation of ubiquitinated proteins in total cell extracts, and also in complex with Cdc48p. These data suggest that Vms1p functions after substrate ubiquitination. In support of this hypothesis, I found that VMS1 mutants show a decrease in the amount of proteasome that handles ubiquitinated substrates and an increase in the amount of free, latent 20S proteasome holoenzyme. This phenomenon is not a result of the altered expression of proteasome components. Additionally, the restoration of ubiquitinated protein accumulation and the distribution of proteasome subtypes to near wild-type levels require the physical interaction between Vms1p and Cdc48p. Furthermore, Cdc48p may be important for recruitment of Vms1p to the proteasome. Using yeast genetics I provide supporting evidence that indicates that Vms1p does not appear to function with various proteasome assembly chaperones. Quantitative mass spectrometry indicates that Cdc48p-associated proteasome is unaffected by loss of VMS1. Together my data indicates that Vms1p functions with Cdc48p to regulate proteasome subtypes that are required to degrade ubiquitinated substrates.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Tran, Joseph Rjrt37@pitt.eduJRT37
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairBrodsky, Jeffrey Ljbrodsky@pitt.eduJBRODSKY
Committee MemberFrizzell, Raymondfrizzell@pitt.eduFRIZZELL
Committee CoChairHughey, Rebeccahugheyr@pitt.eduHUGHEYR
Committee MemberSchmidt, Martin Cmcs2@pitt.eduMCS2
Committee MemberSmithgall, Thomas Etsmithga@pitt.eduTSMITHGA
Date: 8 April 2013
Date Type: Publication
Defense Date: 17 August 2012
Approval Date: 8 April 2013
Submission Date: 17 December 2012
Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
Number of Pages: 219
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Biochemistry and Molecular Genetics
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Protein quality control, protein degradation, Endoplasmic Reticulum Associated Degradation, Ubiquitin Proteasome System
Date Deposited: 08 Apr 2013 11:40
Last Modified: 15 Nov 2016 14:08
URI: http://d-scholarship.pitt.edu/id/eprint/17011

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  • A CDC48 COFACTOR AFFECTS UBIQUITINATED PROTEIN LEVELS, ENDOPLASMIC RETICULUM ASSOCIATED DEGRADATION AND PROTEASOME SUBTYPES. (deposited 08 Apr 2013 11:40) [Currently Displayed]

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