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Title: On the mechanisms of receptor-mediated retention of soluble endoplasmic reticulum resident proteins in eukaryotes
Author: Alvim, Jonas Chaves
ISNI:       0000 0004 7654 7993
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2018
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Accumulation of soluble proteins in the endoplasmic reticulum (ER) of plants is mediated by a protein receptor termed ER RETENTION DEFECTIVE 2 (ERD2). To study the mechanism for protein accumulation in the ER, I have optimized a previously established bioassay using Nicotiana benthamiana protoplasts, which proved to be outstanding. The combined use of gain-of- function assays, complementation assays, anti-sense inhibition and confocal laser scanning microscopy allowed me to show that biologically active fluorescent ERD2 fusions are exclusively detected at the Golgi apparatus and do not show ligand-induced redistribution to the ER. I also show that ERD2 dual ER-Golgi distribution is accompanied to lack-of biological function due to the masking a novel C-terminal di-leucine motif. This motif is shown to not promote rapid ER export, but it prevents recycling from the Golgi apparatus back to the ER. Further analysis revealed that the ERD2 C-terminus is necessary but not sufficient to mediate Golgi residency. ERD2 C-terminus replacement by a canonical KKXX sequence, caused biological inactivity and exclusive ER localisation. Together, the data suggest that Golgi-residency and biological activity are directly linked, which argues strongly against the typical receptor-recycling model that has been accepted for so long. Interestingly, I found an astonishingly high degree of conservation of the receptor amongst eukaryotes, and only the receptor from few species were unable to mediate ER retention of soluble ligands in plants. Combined assays provided the experimental platform to classify further ERD2 mutants into three different functional classes. Finally, I generated further data suggesting the ERD2 may have a role in vacuolar transport and protein turnover. I conclude from my work that the classical recycling model for ERD2-mediated accumulation of soluble proteins in the ER may have to be challenged in the future and that much is to be discovered about the ER-Golgi interface in eukaryotes.
Supervisor: Denecke, Jurgen Sponsor: Brazilian National Council for Scientific and Technological Development (CNPq)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available