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Insight into ER quality control mechanisms : novel characterization of the E3 ubiquitin ligase gp78/autocrine motility factor receptor and the G<alpha>s subunit in the ER Fairbank, Maria


This manuscript looks at ER Quality Control (ERQC) mechanisms and in particular, focuses on two cellular pathways: (1) the ER-associated degradation (ERAD) pathway and (2) the ER stress response. ERQC represents a complex assembly of pathways that are vital in maintaining proper cellular function and homeostasis, by helping the cell adapt to ER stress, prevent chronic imbalance in the ER and avoid many protein conformational diseases. Here, we investigate (1) a regulatory role for palmitoylation of the E3 ubiquitin ligase gp78/AMFR in the ERAD pathway; (2) the implication of G proteins in gp78/AMFR functions; and (3) the involvement of ER-localized Gαs in both substrate polyubiquitylation and ER stress. The dynamic posttranslational modification, palmitoylation, is important for receptor stability and intracellular trafficking. Using metabolic radiolabeling and Acyl-Biotinyl Exchange Chemistry, in chapter 2, we show that the E3 ubiquitin ligase gp78/AMFR is palmitoylated within the catalytic RING finger motif, a domain that is responsible for its ubiquitin ligase activity. We also discuss the modulatory implication of gp78/AMFR palmitoylation, showing that palmitoylation disrupts the RING finger motif, regulates its ER distribution and enhances its turnover. Whether palmitoylation of E3 ubiquitin ligases is gp78/AMFR-specific or a general mechanism to control the activity of RING finger ubiquitin ligases remains to be determined. Next, we look at the Gα subunit, a known component of the G protein-coupled receptor (GPCR) signal transduction pathway. In chapter 3, using immunoprecipitation and immunocytochemistry experiments, we report that the E3 ubiquitin ligase gp78/AMFR interacts with and recruits several G proteins to the ER, namely Gαi1 and Gαs. Thus, we reintroduce the possibility that gp78/AMFR is a novel ER-localized GPCR. The Gαs subunit is further discussed in chapter 4 where we characterize in detail its ER localization and its association with ERAD components, as well as we show a novel intracellular function, demonstrating the ability of Gαs to induce substrate polyubiquitylation and protect against ER stress. Together, these findings mark the beginning in understanding the physiological significance of (1) E3 ubiquitin ligase palmitoylation; (2) G protein binding to gp78/AMFR; (3) Gαs-mediated substrate polyubiquitylation and protection against ER stress, in ERQC mechanisms.

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