Utilizing Drosophila S2 Cells as a Model System to Determine Underlying Mechanisms of ER-associated Degradation
| dc.contributor.advisor | Seemann, Joachim | en |
| dc.contributor.committeeMember | DeBose-Boyd, Russell A. | en |
| dc.contributor.committeeMember | Smith, Dean P. | en |
| dc.contributor.committeeMember | Thomas, Philip J. | en |
| dc.creator | Faulkner, Rebecca Ann | en |
| dc.date.accessioned | 2016-06-27T20:06:07Z | |
| dc.date.available | 2016-06-27T20:06:07Z | |
| dc.date.created | 2014-05 | |
| dc.date.issued | 2014-03-17 | |
| dc.date.submitted | May 2014 | |
| dc.date.updated | 2016-06-27T19:47:55Z | |
| dc.description.abstract | Proper folding of nascent polypeptides is vital to maintain cellular homeostasis; proteins that adopt aberrant confirmations are selectively degraded through a multi-step process known as endoplasmic reticulum-associated degradation (ERAD). Underlying mechanisms for ERAD of membrane proteins, especially those with multiple membrane-spanning segments, are poorly understood. There are currently many unknown aspects of ERAD including mechanisms for selection of polytopic substrates, whether dislocation from ER membranes into the cytosol requires a protein conducting channel, how solubility of the transmembrane domains are maintained during dislocation and delivery to proteasomes for degradation. I have begun to address these questions by examining the ERAD of integral membrane proteins, HMG CoA reductase and Insig-1. These are ideal model substrates since their ERAD is strictly regulated by lipids, which helps guard against artifacts when various aspects of the reactions are reconstituted in model systems or in vitro. I utilized Drosophila S2 cells as a model system to identify proteins required for ERAD and cytosolic dislocation of HMG CoA reductase and Insig-1. S2 cells offer several advantages over mammalian cells, i.e., ease of transgene overexpression, simpler genome, and robust execution of RNA interference. Previously, my laboratory reconstituted sterol-regulated ERAD of mammalian reductase in S2 cells and identified dHrd1 as the ubiquitin ligase required for the reaction. Using tandem affinity purification of dHrd1 and mass spectrometry, I identified components of the Drosophila ERAD pathway that associate with dHrd1. A role for dHrd1-associated proteins in cytosolic dislocation and ERAD of HMG CoA reductase was subsequently established using RNA interference. I also defined a role for dSel1, a dHrd1 complex component, in selection of reductase for ERAD, and identified the region of dSel1 required to bridge reductase to Insig-1. Finally, I demonstrated that physiologic conditions for Insig-1 ERAD in S2 cells are consistent with those in mammalian cells and identified dTeb4 as the ubiquitin ligase required for the reaction. Data indicate dHrd1 and dTeb4 share common ERAD components. Surprisingly, genetic and pharmacologic experiments indicate that Insig-1 and reductase are degraded through distinct mechanisms mediated by different ubiquitin ligase complexes in S2 cells. | en |
| dc.format.mimetype | application/pdf | en |
| dc.identifier.oclc | 952355678 | |
| dc.identifier.uri | https://hdl.handle.net/2152.5/3312 | |
| dc.language.iso | en_US | en |
| dc.subject | Endoplasmic Reticulum-Associated Degradation | en |
| dc.subject | Hydroxymethylglutaryl CoA Reductases | en |
| dc.subject | Intracellular Signaling Peptides and Proteins | en |
| dc.subject | Membrane Proteins | en |
| dc.title | Utilizing Drosophila S2 Cells as a Model System to Determine Underlying Mechanisms of ER-associated Degradation | en |
| dc.type | Thesis | en |
| dc.type.material | text | en |
| thesis.degree.department | Graduate School of Biomedical Sciences | en |
| thesis.degree.discipline | Cell Regulation | en |
| thesis.degree.grantor | UT Southwestern Medical Center | en |
| thesis.degree.level | Doctoral | en |
| thesis.degree.name | Doctor of Philosophy | en |