The vast majority of theses in this collection are open access and freely available. There are a small number of theses that have access restricted to the WWU campus. For off-campus access to a thesis labeled "Campus Only Access," please log in here with your WWU universal ID, or talk to your librarian about requesting the restricted thesis through interlibrary loan.

Date Permissions Signed

8-13-2020

Date of Award

Summer 2020

Document Type

Masters Thesis

Department or Program Affiliation

Biology

Degree Name

Master of Science (MS)

Department

Biology

First Advisor

Dahlberg, Caroline Lund, 1978-

Second Advisor

Rose, Jacqueline K.

Third Advisor

Serrano-Moreno, Jose Ramon

Abstract

Neurons communicate with other cells to elicit outputs that include memory and movement. Cells, including neurons, create proteins every day for specific functions and in particular, neurons produce proteins that enable their communication. Proteins found in cellular membranes are synthesized at the endoplasmic reticulum (ER). However, up to 30% of new proteins are improperly folded and must be removed from the cell. A build-up of misfolded proteins can trigger the Unfolded Protein Response (UPR) which initiates other pathways of protein quality control and can determine the fate of a cell. ER-associated protein degradation (ERAD) is a ubiquitin-dependent process in eukaryotic cells that helps alleviate protein accumulation by breaking down misfolded proteins and targeting them to for degradation at the proteasome. The ERAD system is well described in yeast but is less well studied in multicellular systems. We used Caenorhabditis elegans (C. elegans) as a model organism to examine the trafficking of a single transmembrane protein through the ER under stressed conditions and as part of the ERAD pathway. The glutamate receptor, GLR-1, is a model membrane protein expressed in a subset of interneurons in C. elegans at the ventral nerve cord (VNC) to observe if GLR-1 is a candidate substrate for ERAD. Assessing GLR-1’s accumulation in the absence of ERAD E3 ubiquitin ligases in C. elegans can determine the protein’s regulation in ERAD. Three putative ERAD E3 ligases in C. elegans are: HRD-1, MARC-6, and HRDL-1. Previous results showed that deletion of most of the gene encoding HRDL-1, caused an increased accumulation of GLR-1::GFP in the VNC. HRDL-1 may have a role in regulating GLR-1 degradation at the ER and GLR-1 may be a candidate endogenous substrate for ERAD. In order to understand how ERAD effects GLR-1, we used a chemical stressor, tunicamycin, which blocks ER-dependent glycosylation and initiates the UPR. Tunicamycin treatment induces ER stress, which activates a UPR-reporter construct hsp-4p::GFP in C. elegans. The hsp4p::GFP reporter responds to tunicamycin at lower doses when animals harbor the mutation gene hrdl-1. We used quantitative fluorescence imaging and immunoblotting to determine whether the GLR-1::GFP accumulated due to underlying ER stress caused by the lack of HRDL-1. Contrary to previous results, we found no difference in fluorescently-tagged GLR-1 in the VNC in animals lacking hrdl-1, though overall levels of tagged GLR-1 were increased. GLR-1 with a point mutation that caused ER accumulation was not affected by the loss of hrdl-1. We propose that another ER E3 ligase may be involved in GLR-1’s regulation in the VNC that further investigation of cell bodies may uncover mechanisms of GLR-1 regulation at the ER.

Type

Text

Keywords

Glutamate, Endoplasmic reticulum, Caenorhabditis elegans, ubiquitin

Publisher

Western Washington University

OCLC Number

1190759509

Subject – LCSH

Glutamic acid--Receptors; Endoplasmic reticulum; Caenorhabditis elegans; Proteolysis

Format

application/pdf

Genre/Form

masters theses

Language

English

Rights

Copying of this document in whole or in part is allowable only for scholarly purposes. It is understood, however, that any copying or publication of this document for commercial purposes, or for financial gain, shall not be allowed without the author’s written permission.

Rights Statement

http://rightsstatements.org/vocab/InC/1.0/

Download

Included in

Biology Commons

Share

COinS