Preston, George
(2017)
SUBSTRATE INSOLUBILITY DICTATES HSP104-DEPENDENTENDOPLASMIC RETICULUM ASSOCIATED DEGRADATION.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
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
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| Status: |
Unpublished |
| Creators/Authors: |
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| ETD Committee: |
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| 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: |
αENAC, 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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