Novel use of glycosylation scanning to map the intracellular trafficking of sarco(endo)plasmic reticulum calcium ATPase 1A
| Author(s) | Flinn, Rory J. | |
| Date Accessioned | 2020-07-27T11:41:59Z | |
| Date Available | 2020-07-27T11:41:59Z | |
| Publication Date | 2005 | |
| Abstract | The sarco(endo)plasmic reticulum calcium ATPase (SERCA) family of proteins function as calcium pumps in the endoplasmic reticulum (ER) and sarcoplasmic reticulum (SR) membranes. SERCA1a is found exclusively in fast-twitch muscle cells and mediates muscle relaxation by pumping calcium back into the SR after calcium has been released into the cytoplasm to elicit muscle contraction. The mechanism which allows SR biogenesis is not known, but SR membrane is believed to bud from the ER. One hypothesis is that SERCA1a proteins play a significant role in SR biogenesis in fast-twitch skeletal muscle due the protein’s large size and clustering into large arrays in the SR membrane. SERCA1a arrays could recruit lipids which would allow for a large increase in membrane size that could result in the formation of the SR. Also, SERCA1a is highly expressed during the early stages of myogenesis, at the same time the first emergence of the SR is observed. It is known that SERCA1a contains ER targeting information and is synthesized in the ER membrane, however the intracellular trafficking pattern of SERCA1a before entering the SR membrane is unknown. Glycosylation events differ between glycoproteins retained in the ER and glycoproteins having traversed some or all of the Golgi apparatus compartments such that a glycoprotein can be analyzed for the particular sugar residues it contains to determine which cellular compartments it has traversed. It was found that calsequestrin, a soluble glycoprotein found in the lumen of the SR, traveled through the Golgi apparatus and arrived in the SR lumen via clathrin-coated vesicles. These vesicles did not contain SERCA1a, and since SERCA1a and calsequestrin are synthesized at overlapping time periods in myogenesis, the hypothesis can be made that SERCA1a does not travel through the Golgi apparatus before entering the SR lumen. It is our hypothesis that SERCA1a is retained in the ER lumen and plays a significant role in SR biogenesis. In order to validate this hypothesis the intracellular trafficking of SERCA1a was investigated. Unlike calsequestrin, SERCA1a is not a glycoprotein. A novel application of a technique called glycosylation scanning was undertaken to create mutant SERCA1a glycoproteins in mouse fibroblasts and mouse myoblasts that were then analyzed for their glycosylation patterns to determine the particular cellular compartments the mutant SERCA1a proteins had traveled through. Three such mutant SERCA1a cDNA constructs were created and used in transfection experiments to generate mutant SERCA1a proteins. Only one of these three glycosylation mutants had expression levels sufficient for further analysis. However, it was concluded that the mutant SERCA1a protein was not glycosylated, and thus no conclusions could be made about the intracellular trafficking of SERCA1a. | en_US |
| Advisor | Karin, Norman J. | |
| Degree | M.S. | |
| Department | University of Delaware, Department of Biological Sciences | |
| Unique Identifier | 73902075 | |
| URL | http://udspace.udel.edu/handle/19716/27348 | |
| Publisher | University of Delaware | en_US |
| URI | https://search.proquest.com/docview/304992777?accountid=10457 | |
| dc.subject.lcsh | Sarcoplasmic reticulum | |
| dc.subject.lcsh | Endoplasmic reticulum | |
| dc.subject.lcsh | Muscle contraction -- Regulation | |
| dc.subject.lcsh | Calcium -- Metabolism | |
| dc.subject.lcsh | Calcium-binding proteins | |
| dc.subject.lcsh | Glycosylation | |
| Title | Novel use of glycosylation scanning to map the intracellular trafficking of sarco(endo)plasmic reticulum calcium ATPase 1A | en_US |
| Type | Thesis | en_US |