The activity of a human endoplasmic reticulum-associated degradation E3, gp78, requires its Cue domain, RING finger, and an E2-binding site

Chen, Bin; Mariano, Jennifer; Tsai, Ya Chea; Chan, H. S. O.; Cohen, Mickaël M.; Weissman, Allan M.

doi:10.1073/pnas.0506618103

Cited by 193 publications

(232 citation statements)

References 40 publications

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“…Coexpression of wild-type gp78-Myc had no effect on the degradation of CFTR⌬F508-GFP (Supplemental Figure 3), even though it enhanced the ubiquitylation of CFTR⌬F508-GFP. Overexpression of gp78 has been shown to accelerate the degradation of other gp78 substrates Song et al, 2005;Chen et al, 2006), which suggests that additional factor(s) are required for the degradation of CFTR⌬F508. HEK293 cells transiently expressing CFTR⌬F508-GFP and HA-ubiquitin, with or without (control lane) the indicated ubiquitin ligases, were lysed and subjected to immunoprecipitation by using anti-GFP antibody, followed by immunoblot using anti-HA antibody.…”

Section: Gp78 Is Involved In Erad Of Cftr⌬f508mentioning

confidence: 99%

“…The association of gp78 with CFTR⌬F508 was mediated by its intrinsic CUE domain, which also has ubiquitin binding activity (Supplemental Figure 4; Zhong et al, 2004;Chen et al, 2006). To determine whether the ubiquitin binding region of the CUE domain overlapped the CFTR⌬F508 binding region, we generated a gp78 mutant in which the ubiquitin binding site in the CUE domain, MFP (amino acids 463-465), was mutated to AAR.…”

Section: The Cue Domain Of Gp78 Is Required For Cftr⌬f508 Bindingmentioning

confidence: 99%

“…Similar to Hrd1p, gp78 is a RING finger-dependent ubiquitin ligase, an integral ER membrane protein, and it is involved in ERAD, in cooperation with Ube2g2/UBC7, the mammalian counterpart of yeast Ubc7p (Fang et al, 2001;Liang et al, 2003;Song et al, 2005;Lee et al, 2006). Recently, Chen et al (2006) demonstrated that both the E2 binding site and the coupling of ubiquitin to ER degradation (CUE) domain of gp78 are essential for its ubiquitin ligase activity. Although recent biochemical and structural studies have helped define the ubiquitin binding properties of CUE domains (Ponting, 2000;Kang et al, 2003;Prag et al, 2003), the role of the gp78 CUE domain in ubiquitin ligation remains unclear.…”

Section: Introductionmentioning

confidence: 99%

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Gp78 Cooperates with RMA1 in Endoplasmic Reticulum-associated Degradation of CFTRΔF508

Morito

Hirao

Oda

et al. 2008

MBoC

211

213

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Misfolded or improperly assembled proteins in the endoplasmic reticulum (ER) are exported into the cytosol and degraded via the ubiquitin-proteasome pathway, a process termed ER-associated degradation (ERAD). Saccharomyces cerevisiae Hrd1p/Der3p is an ER membrane-spanning ubiquitin ligase that participates in ERAD of the cystic fibrosis transmembrane conductance regulator (CFTR) when CFTR is exogenously expressed in yeast cells. Two mammalian orthologues of yeast Hrd1p/Der3p, gp78 and HRD1, have been reported. Here, we demonstrate that gp78, but not HRD1, participates in ERAD of the CFTR mutant CFTR⌬F508, by specifically promoting ubiquitylation of CFTR⌬F508. Domain swapping experiments and deletion analysis revealed that gp78 binds to CFTR⌬F508 through its ubiquitin binding region, the so-called coupling of ubiquitin to ER degradation (CUE) domain. Gp78 polyubiquitylated in vitro an N-terminal ubiquitin-glutathione-S-transferase (GST)-fusion protein, but not GST alone. This suggests that gp78 recognizes the ubiquitin that is already conjugated to CFTR⌬F508 and catalyzes further polyubiquitylation of CFTR⌬F508 in a manner similar to that of a multiubiquitin chain assembly factor (E4). Furthermore, we revealed by small interfering RNA methods that the ubiquitin ligase RMA1 functioned as an E3 enzyme upstream of gp78. Our data demonstrates that gp78 cooperates with RMA1 with E4-like activity in the ERAD of CFTR⌬F508.

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Section: Gp78 Is Involved In Erad Of Cftr⌬f508mentioning

confidence: 99%

Section: The Cue Domain Of Gp78 Is Required For Cftr⌬f508 Bindingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Gp78 Cooperates with RMA1 in Endoplasmic Reticulum-associated Degradation of CFTRΔF508

Morito

Hirao

Oda

et al. 2008

MBoC

211

213

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“…RING finger E3s usually interact with cognate E2s via the RING domain. However, gp78 was reported to contain a second E2 binding site named G2BR (24). To understand how gp78 interacts with Ube2g2 to assemble a functional E3-E2 heterooligomer, we determined the crystal structure of Ube2g2 bound to a 28-residue peptide corresponding to the G2BR domain of gp78.…”

Section: Structure Of Ube2g2mentioning

confidence: 99%

“…We also showed that gp78 forms an oligomer that might be critical for E2 active site-associated polyubiquitination (16). gp78 is a multispanning membrane protein anchored to the ER membrane with its catalytic domain facing the cytosol (23,24). To identify the domains responsible for gp78 oligomerization, we expressed Flag-tagged, full-length gp78 (Flaggp78) together with GFP-fusion proteins comprising either the N-terminal transmembrane segments (gp78N-GFP) or the Cterminal cytosolic domain (gp78C-GFP) of gp78 in cells.…”

mentioning

confidence: 99%

Mechanistic insights into active site-associated polyubiquitination by the ubiquitin-conjugating enzyme Ube2g2

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

115

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Lys-48-linked polyubiquitination regulates a variety of cellular processes by targeting ubiquitinated proteins to the proteasome for degradation. Although polyubiquitination had been presumed to occur by transferring ubiquitin molecules, one at a time, from an E2 active site to a substrate, we recently showed that the endoplasmic reticulum-associated RING finger ubiquitin ligase gp78 can mediate the preassembly of Lys-48-linked polyubiquitin chains on the catalytic cysteine of its cognate E2 Ube2g2 and subsequent transfer to a substrate. Active site-linked polyubiquitin chains are detected in cells on Ube2g2 and its yeast homolog Ubc7p, but how these chains are assembled is unclear. Here, we show that gp78 forms an oligomer via 2 oligomerization sites, one of which is a hydrophobic segment located in the gp78 cytosolic domain. We further demonstrate that a gp78 oligomer can simultaneously associate with multiple Ube2g2 molecules. This interaction is mediated by a novel Ube2g2 surface distinct from the predicted RING binding site. Our data suggest that a large gp78 -Ube2g2 heterooligomer brings multiple Ube2g2 molecules into close proximity, allowing ubiquitin moieties to be transferred between neighboring Ube2g2s to form active site-linked polyubiquitin chains.crystallography ͉ endoplasmic reticulum-associated protein degradation ͉ gp78 ͉ polyubiquitin chain C ovalent attachment of the 76-residue ubiquitin to polypeptides regulates the stability, localization, or activity of the modified proteins (1-3). This modification requires concerted actions of 3 types of enzymes: an activating enzyme (E1) that forms a thioester linkage (designated herein as ''ϳ'') between its catalytic cysteine and the carboxyl group of the Gly-76 in ubiquitin; a conjugating enzyme (E2) that receives ubiquitin from an E1; and an ubiquitin ligase (E3) that catalyzes the transfer of ubiquitin from the E2 active-site cysteine to a substrate (4). To date, 2 E1s and dozens of E2 enzymes have been identified in mammals, whereas the number of E3s is in the range of several hundreds (5-8). Many E3 enzymes contain a RING finger domain that interacts transiently with a cognate E2 to mediate polyubiquitination reactions (4, 9-12).Ubiquitination often occurs in the form of a polymer whereby the C-terminal Gly-76 in an ubiquitin moiety is linked to a lysine residue in another ubiquitin molecule. It had been presumed that polyubiquitin chains are formed by conjugating ubiquitin moieties, one at a time, first to a lysine residue in a substrate, and then to a lysine in the previously-attached ubiquitin molecule. This appears to be the case for some E2s (13-15). However, we and several other groups recently found that ubiquitin chains can also be preassembled on the catalytic cysteine of the E2 enzyme Ube2g2 and its yeast homologue Ubc7p both in vitro and in vivo (16)(17)(18)(19). These ubiquitin-conjugating enzymes are associated with endoplasmic reticulum (ER) membranes to modify misfolded polypeptides that have been exported from the ER lumen in a proc...

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Endoplasmic Reticulum Protein Quality Control and Degradation

Schfer¹,

Kostova²,

Wolf³

2007

Protein Degradation Series

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The activity of a human endoplasmic reticulum-associated degradation E3, gp78, requires its Cue domain, RING finger, and an E2-binding site

Cited by 193 publications

References 40 publications

Gp78 Cooperates with RMA1 in Endoplasmic Reticulum-associated Degradation of CFTRΔF508

Gp78 Cooperates with RMA1 in Endoplasmic Reticulum-associated Degradation of CFTRΔF508

Mechanistic insights into active site-associated polyubiquitination by the ubiquitin-conjugating enzyme Ube2g2

Endoplasmic Reticulum Protein Quality Control and Degradation

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