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Functional roles of ubiquitin ligase gp78 in endoplasmic reticulum domains

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Title: Functional roles of ubiquitin ligase gp78 in endoplasmic reticulum domains
Author: St-Pierre, Pascal
Degree Doctor of Philosophy - PhD
Program Cell and Developmental Biology
Copyright Date: 2012
Publicly Available in cIRcle 2012-09-12
Abstract: The ubiquitin ligase gp78, involved in ER-associated degradation, has been extensively studied using the 3F3A monoclonal antibody, and correlates with increased metastasis incidence in cancer patients (review in Chiu et al. 2008) The 3F3A-labeled ER characterized as an ER domain in close association with mitochondria distinct of the reticulon-labeled ER tubules, the juxtanuclear and the perinuclear ER. Overexpression of gp78 saw 3F3A expansion to the peripheral ER, but remained in association with mitochondria. This association is calcium-sensitive as elevation in cytosolic calcium levels disrupted contact with mitochondria. Similarly, induction of calcium released from the ER through thapsigargin or ATP stimulation of purinegic receptors promoted dissociation of 3F3A labeled ER and mitochondria. Upon ER-mitochondria dissociation, the IP3R-labeled ER dissociated in a distinct domain from the 3F3A-labeled ER, indicating that regulation of the ER-mitochondria association by free cytosolic calcium is a characteristic of smooth ER domains and that multiple mechanisms regulate the interactions between these organelles. Upon overexpression of gp78, the 3F3A labeled peripheral ER was identified as the site of gp78-mediated ubiquitylation. Gp78 polyubiquitylated substrates where stabilized through Cue domain interaction in the peripheral ER. Derlin-1 and derlin-2, involved in retrotranslocation of ERAD substrates, localized to a juxtanuclear ER domain, where ubiquitylated proteins accumulate upon proteasome inhibition. This segregation was shown in a non-overexpressing system using HT-1080 fibrosarcoma cells expressing elevated levels of endogenous gp78. This shows the special segraqgation of ERAD by gp78-mediatedubiquitin ligase activity in the peripheral ER. This may function in ER quality control and regulate the expression of smooth ER-associated substrates in response to physiological change. Finally, we found that stimulation of gp78 at the cell surface by its ligand AMF disrupts ER-mitochondria interaction, calcium coupling and decreases gp78 ubiquitin ligase activity. Disruption of the ubiquitin ligase activity via mutation of gp78 RING domain or expression of ubiquitin mutant unable to form elongated polyubiquitin chains disrupts ER-mitochondria coupling. The increased fragmentation and reduced mobility of mitochondria upon gp78 overexpression were dependant on ubiquitin ligase activity and prevented by AMF treatment. These results describe a novel mechanism regulating mitochondrial dynamics via the ER ubiquitin ligase gp78.
URI: http://hdl.handle.net/2429/43202
Scholarly Level: Graduate

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