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Title: Elucidating the recycling mechanism of ER resident proteins with ERD2
Author: An, Jing
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2015
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One of the key steps for maintaining the endoplasmic reticulum (ER)-Golgi interface in the early secretory pathway is the receptor-mediated recycling of abundant soluble ER residents containing K/HDEL signals from the Golgi apparatus. The receptor was identified in yeast by screening for ER retention defective (ERD) mutants, and the ERD2 gene product was shown to mediate both the specificity and the capacity of ER retention in yeast. Since its discovery in 1990, ERD2 homologues have been identified from numerous eukaryotes including plants, and it is currently believed that it directly regulates the formation of retrograde transport vesicles from the Golgi. However, it is not understood how it recycles efficiently between the ER and the Golgi, and how it avoids leakage from the Golgi to later compartments. Although the recycling principle has been used as an argument to explain how few receptors could retain many ligands in the ER, it is unclear how receptors recycle to the Golgi faster than the unspecific anterograde bulk flow. Here I establish a quantitative receptor-ligand interaction study to determine the receptor to ligand ratio in vivo. Using the model cargo α-amylase-HDEL (Amy-HDEL) and co-expressed ERD2 I succeeded in up regulating the retention capacity of plant cells. This bioassay has been developed into a quantitative method using internal reference markers for normalising gene transfer, and tagging of receptors and ligands to permit simultaneous detection. Results are complemented by confocal laser scanning microscopy (CLSM) which demonstrates the ERD2 mediated ER retention. The activity assay was also used to identify critical amino acids in ERD2 function. Unexpectedly, the cytosolic C-terminus was found to be essential in maintaining the ability of ERD2 to retain HDEL proteins, in contrast to previous studies based on in vitro peptide binding assays. In addition to loss-of-function mutations, I also identified a new class of ERD2 mutant that causes induced secretion of HDEL proteins. These mutant classes are currently tested with respect to the sorting characteristics of the receptor, to distinguish between ligand-binding mutants and protein sorting mutants.
Supervisor: Denecke, Jurgen Sponsor: Zhenya Zhang Foundation
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available