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SUBSTRATE INSOLUBILITY DICTATES HSP104-DEPENDENTENDOPLASMIC RETICULUM ASSOCIATED DEGRADATION

Preston, George (2017) SUBSTRATE INSOLUBILITY DICTATES HSP104-DEPENDENTENDOPLASMIC RETICULUM ASSOCIATED DEGRADATION. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

Misfolded proteins that reside in the Endoplasmic Reticulum (ER) are degraded through a quality control process known as ER-associated degradation (ERAD). ERAD substrates are targeted to the 26S proteasome, but when proteasome function is compromised disease-associated ERAD substrates can aggregate in the cytoplasm. Under normal conditions, how are these misfolded substrates identified by ERAD factors? To address this question, a novel ERAD substrate, Q394X, was utilized. Q394X contains two transmembrane domains fused to a truncated, C-terminal Nucleotide Binding Domain (NBD) from the well-characterized ERAD substrate, Ste6p*. By exposing ER-enriched microsomes containing Q394X to the non-ionic detergent, dodecyl maltoside (DDM), Q394X was shown to be partially extracted from membranes and is less soluble than the wild type version of Q394X (Chimera A). However, Q394X solubility was restored after addition of 6M urea to similar levels as Chimera A. Interestingly, systematically truncating the NBD at sites both N-terminal and C-terminal to the truncation site in Q394X had varying effects on protein solubility and stability. Two of the truncation mutants were as soluble as Chimera A, while the others exhibited the decreased solubility that was evident for Q394X. By performing degradation assays, it was demonstrated that protein solubility and stability are positively correlated.
To define how cells process the less soluble substrate, Q394X, I next investigated which chaperones might mediate its degradation. I found that Q394X was stabilized in yeast when genes encoding members of the protein disaggregation machinery, including the AAA+ ATPase, Heat
SUBSTRATE INSOLUBILITY DICTATES HSP104-DEPENDENT
ENDOPLASMIC RETICULUM ASSOCIATED DEGRADATION
George Michael Preston, Ph.D.
University of Pittsburgh, 2017
v
Shock Protein of 104 kDa (Hsp104), were deleted. Under these conditions of Q394X stabilization in the absence of Hsp104, I determined that Q394X aggregates in the ER membrane. Furthermore, Hsp104 was shown to localize to punctae under these conditions, but formation of these punctae requires Q394X expression. This aggregation of Q394X leads to a decrease in Q394X retrotranslocation by Cdc48. These data provide evidence that Hsp104 acts to disaggregate Q394X in the ER membrane, which is required for efficient Q394X retrotranslocation and degradation.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Preston, Georgegmp26@gmail.comgmp26
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Thesis AdvisorBrodsky, Jeffreyjbrodsky@pitt.edujbrodsky
Committee ChairThibodeau, Patrickthibodea@pitt.eduthibodea
Committee MemberSubramanya, Arohanars129@pitt.eduars129
Committee MemberWan, Yongyow4@pitt.eduyow4
Committee MemberVanDemark, Andrewandyv@pitt.eduandyv
Date: 5 July 2017
Date Type: Publication
Defense Date: 13 April 2017
Approval Date: 5 July 2017
Submission Date: 8 June 2017
Access Restriction: 1 year -- Restrict access to University of Pittsburgh for a period of 1 year.
Number of Pages: 158
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Cell Biology and Molecular Physiology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: ╬▒ENAC, The alpha subunit of the heterotrimeric epithelial sodium channel CF, Cystic Fibrosis CFTR, cystic fibrosis transmembrane conductance regulator CHIP, C-terminus of Hsc70- interacting protein DDM, non-ionic detergent, dodecyl maltoside DUBs, deubiquitinating enzymes ER, Endoplasmic Reticulum ERAD, Endoplasmic reticulum-associated degradation ERAD-C, ER cytosolic lesion ERAD-L, ER luminal lesion HECT, homologous to the E6AP carboxyl terminus HRD, Hmg CoA reductase degradation HSP, heat-shock protein Q394X, Chimera AQ394X RING, really interesting new gene SCF, Skp1, Cullins, F-box proteins SUMO, small ubiquitin-like modifier UBX, ubiquitin regulatory X UPS, ubiquitin-proteasome system
Date Deposited: 05 Jul 2017 19:59
Last Modified: 05 Jul 2018 05:15
URI: http://d-scholarship.pitt.edu/id/eprint/32416

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