Peripheral Endoplasmic Reticulum Localization of Gp78 Ubiquitin Ligase Activity

St-Pierre, Pascal; Dang, Thao Thi Phuong; Joshi, Bharat; Nabi, Ivan R.

doi:10.1242/jcs.096396

Cited by 23 publications

(29 citation statements)

References 51 publications

(70 reference statements)

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“…1C). Similarly, co-transfection of FlagGp78wt with mutated HA-tagged ubiquitin (K29R, K48R and K63R, denoted HA-Ubmono), preventing Gp78-dependent polyubiquitin chain elongation (St-Pierre et al, 2012), results in longer and AMF-insensitive coupling times (Fig. 1C).…”

Section: Results and Discussion Amf-gp78 Regulation Of Er-mitochondrimentioning

confidence: 90%

“…To study Gp78-mediated regulation of ER-mitochondria interaction by electron microscopy, we stably transfected HT-1080 fibrosarcoma cells, which express elevated levels of endogenous Gp78 (St-Pierre et al, 2012;Tsai et al, 2007), with doxycyclininducible Gp78 short hairpin RNA (shRNA; shGp78). 3D confocal analysis showed that shGp78 knockdown reduced syntaxin-17 overlap with mitochondria ( Fig.…”

Section: Amf-gp78 Selectively Regulate Rer-mitochondria Contactsmentioning

confidence: 99%

“…Peripheral ER 3F3A labeling identified Flag-Gp78-transfected cells (St-Pierre et al, 2012). HT-1080 cells (expressing RFP) were labeled with mouse OxPhosV mAb and rabbit Syntaxin17 antibodies and secondary antibodies conjugated to Alexa Fluor 488 and 647.…”

Section: Immunofluorescencementioning

confidence: 99%

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Distinct mechanisms controlling rough and smooth endoplasmic reticulum-mitochondria contacts

Wang

Garcin

et al. 2015

Journal of Cell Science

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102

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Gp78 (also known as AMFR), an endoplasmic-reticulum (ER)-associated protein degradation (ERAD) E3 ubiquitin ligase, localizes to mitochondria-associated ER and targets the mitofusin (Mfn1 and Mfn2) mitochondrial fusion proteins for degradation. Gp78 is also the cell surface receptor for autocrine motility factor (AMF), which prevents Gp78-dependent mitofusin degradation. Gp78 ubiquitin ligase activity promotes ER-mitochondria association and ER-mitochondria Ca 2+ coupling, processes that are reversed by AMF. Electron microscopy of HT-1080 fibrosarcoma cancer cells identified both smooth ER (SER; ∼8 nm) and wider (∼50-60 nm) rough ER (RER)-mitochondria contacts. Both short hairpin RNA (shRNA)-mediated knockdown of Gp78 (shGp78) and AMF treatment selectively reduced the extent of RER-mitochondria contacts without impacting on SER-mitochondria contacts. Concomitant small interfering RNA (siRNA)-mediated knockdown of Mfn1 increased SER-mitochondria contacts in both control and shGp78 cells, whereas knockdown of Mfn2 increased RER-mitochondria contacts selectively in shGp78 HT-1080 cells. The mitofusins therefore inhibit ER-mitochondria interaction. Regulation of close SER-mitochondria contacts by Mfn1 and of RER-mitochondria contacts by AMFsensitive Gp78-mediated degradation of Mfn2 define new mechanisms that regulate ER-mitochondria interactions.

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Section: Results and Discussion Amf-gp78 Regulation Of Er-mitochondrimentioning

confidence: 90%

Section: Amf-gp78 Selectively Regulate Rer-mitochondria Contactsmentioning

confidence: 99%

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Distinct mechanisms controlling rough and smooth endoplasmic reticulum-mitochondria contacts

Wang

Garcin

et al. 2015

Journal of Cell Science

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102

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“…Doxycyclin inducible Gp78-targeted shRNA lentiviral vectors incorporating two shRNAs (sh2 and sh6) that effectively targeted Gp78 (St-Pierre et al, 2012) were used to generate Gp78 knockdown HT-1080 cells. Doxycyclin treatment resulted in reduced Gp78 expression by western blot that was generally more pronounced in sh6 transfected cells ( Fig.…”

Section: Dynamin-dependent Internalization Of Amf Downregulates Its Cmentioning

confidence: 99%

“…Gp78 internalizes AMF via a noncaveolar, dynamin-and PI3K-dependent raft-mediated endocytic pathway to the smooth ER (Benlimame et al, 1998;Kojic et al, 2007;Kojic et al, 2008;Le et al, 2002;Le and Nabi, 2003). We recently localized the initiation of Gp78-dependent substrate ubiquitylation to the peripheral smooth ER domain (St-Pierre et al, 2012). However, the relationship between the raftdependent endocytosis of AMF, its signaling and Gp78 function in ERAD remains unknown.…”

Section: Introductionmentioning

confidence: 99%

Raft endocytosis of autocrine motility factor regulates mitochondrial dynamics via rac1 signaling and the gp78 ubiquitin ligase

Shankar

Kojic

St-Pierre

et al. 2013

Journal of Cell Science

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SummaryGp78 is a cell surface receptor that also functions as an E3 ubiquitin ligase in the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. The Gp78 ligand, the glycolytic enzyme phosphoglucose isomerase (PGI; also called autocrine motility factor, AMF), functions as a cytokine upon secretion by tumor cells. AMF is internalized through a PI3K-and dynamin-dependent raft endocytic pathway to the smooth ER; however, the relationship between AMF and Gp78 ubiquitin ligase activity remains unclear. AMF uptake to the smooth ER is inhibited by the dynamin inhibitor, dynasore, is reduced in Gp78 knockdown cells and induces the dynamin-dependent downregulation of its cell surface receptor. AMF uptake is Rac1-dependent and is inhibited by expression of dominant-negative Rac1 and the Rac1 inhibitor NSC23766, and is therefore distinct from Cdc42-and RhoA-dependent raft endocytic pathways. AMF stimulates Rac1 activation, but this is reduced by dynasore treatment and is absent in Gp78-knockdown cells; therefore, AMF activities require Gp78-mediated endocytosis. AMF also prevents Gp78-induced degradation of the mitochondrial fusion proteins, mitofusin 1 and 2 in a dynamin-, Rac1-and phosphoinositide 3-kinase (PI3K)-dependent manner. Gp78 induces mitochondrial clustering and fission in a manner dependent on GP78 ubiquitin ligase activity, and this is also reversed by uptake of AMF. The raft-dependent endocytosis of AMF, therefore, promotes Rac1-PI3K signaling that feeds back to promote AMF endocytosis and also inhibits the ability of Gp78 to target the mitofusins for degradation, thereby preventing Gp78-dependent mitochondrial fission. Through regulation of an ER-localized ubiquitin ligase, the raft-dependent endocytosis of AMF represents an extracellular regulator of mitochondrial fusion and dynamics.

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Basal Gp78-dependent mitophagy promotes mitochondrial health and limits mitochondrial ROS

Alan

Vandevoorde

Joshi

et al. 2022

Cell. Mol. Life Sci.

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Mitochondria are major sources of cytotoxic reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, that when uncontrolled contribute to cancer progression. Maintaining a finely tuned, healthy mitochondrial population is essential for cellular homeostasis and survival. Mitophagy, the selective elimination of mitochondria by autophagy, monitors and maintains mitochondrial health and integrity, eliminating damaged ROS-producing mitochondria. However, mechanisms underlying mitophagic control of mitochondrial homeostasis under basal conditions remain poorly understood. E3 ubiquitin ligase Gp78 is an endoplasmic reticulum membrane protein that induces mitochondrial fission and mitophagy of depolarized mitochondria. Here, we report that CRISPR/Cas9 knockout of Gp78 in HT-1080 fibrosarcoma cells increased mitochondrial volume, elevated ROS production and rendered cells resistant to carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-induced mitophagy. These effects were phenocopied by knockdown of the essential autophagy protein ATG5 in wild-type HT-1080 cells. Use of the mito-Keima mitophagy probe confirmed that Gp78 promoted both basal and damage-induced mitophagy. Application of a spot detection algorithm (SPECHT) to GFP-mRFP tandem fluorescent-tagged LC3 (tfLC3)-positive autophagosomes reported elevated autophagosomal maturation in wild-type HT-1080 cells relative to Gp78 knockout cells, predominantly in proximity to mitochondria. Mitophagy inhibition by either Gp78 knockout or ATG5 knockdown reduced mitochondrial potential and increased mitochondrial ROS. Live cell analysis of tfLC3 in HT-1080 cells showed the preferential association of autophagosomes with mitochondria of reduced potential. Xenograft tumors of HT-1080 knockout cells show increased labeling for mitochondria and the cell proliferation marker Ki67 and reduced labeling for the TUNEL cell death reporter. Basal Gp78-dependent mitophagic flux is, therefore, selectively associated with reduced potential mitochondria promoting maintenance of a healthy mitochondrial population, limiting ROS production and tumor cell proliferation.

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Peripheral Endoplasmic Reticulum Localization of Gp78 Ubiquitin Ligase Activity

Cited by 23 publications

References 51 publications

Distinct mechanisms controlling rough and smooth endoplasmic reticulum-mitochondria contacts

Distinct mechanisms controlling rough and smooth endoplasmic reticulum-mitochondria contacts

Raft endocytosis of autocrine motility factor regulates mitochondrial dynamics via rac1 signaling and the gp78 ubiquitin ligase

Basal Gp78-dependent mitophagy promotes mitochondrial health and limits mitochondrial ROS

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