Hrd1p/Der3p is a membrane-anchored ubiquitin ligase required for ER-associated degradation

In eukaryotes, endoplasmic reticulum-associated degradation (ERAD) functions in cellular quality control and regulation of normal ER-resident proteins. ERAD proceeds by the ubiquitin-proteasome pathway, in which the covalent attachment of ubiquitin to proteins targets them for proteasomal degradation. Ubiquitin-protein ligases (E3s) play a crucial role in this process by recognizing target proteins and initiating their ubiquitination. Here we show that Hrd1p, which is identical to Der3p, is an E3 for ERAD. Hrd1p is required for the degradation and ubiquitination of several ERAD substrates and physically associates with relevant ubiquitin-conjugating enzymes (E2s). A soluble Hrd1 fusion protein shows E3 activity in vitro - catalysing the ubiquitination of itself and test proteins. In this capacity, Hrd1p has an apparent preference for misfolded proteins. We also show that Hrd1p functions as an E3 in vivo, using only Ubc7p or Ubc1p to specifically program the ubiquitination of ERAD substrates.     

Human HRD1 is an E3 ubiquitin ligase involved in degradation of proteins from the endoplasmic reticulum

The ubiquitin system plays an important role in endoplasmic reticulum (ER)-associated degradation of proteins that are misfolded, that fail to associate with their oligomerization partners, or whose levels are metabolically regulated. E3 ubiquitin ligases are key enzymes in the ubiquitination process as they recognize the substrate and facilitate coupling of multiple ubiquitin units to the protein that is to be degraded. The Saccharomyces cerevisiae ER-resident E3 ligase Hrd1p/Der3p functions in the metabolically regulated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and additionally facilitates the degradation of a number of misfolded proteins from the ER. In this study we characterized the structure and function of the putative human orthologue of yeast Hrd1p/Der3p, designated human HRD1. We show that human HRD1 is a non-glycosylated, stable ER protein with a cytosolic RING-H2 finger domain. In the presence of the ubiquitin-conjugating enzyme UBC7, the RING-H2 finger has in vitro ubiquitination activity for Lys(48)-specific polyubiquitin linkage, suggesting that human HRD1 is an E3 ubiquitin ligase involved in protein degradation. Human HRD1 appears to be involved in the basal degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase but not in the degradation that is regulated by sterols. Additionally we show that human HRD1 is involved in the elimination of two model ER-associated degradation substrates, TCR-alpha and CD3-delta.     

Der1p, a protein required for degradation of malfolded soluble proteins of the endoplasmic reticulum: topology and Der1-like proteins

The endoplasmic reticulum (ER) contains a highly effective protein quality control system eliminating malfolded proteins by a mechanism called ER-associated protein degradation (ERAD). Here, we unravel the topology of Der1p, a previously identified component of the ERAD system. Der1p contains four transmembrane domains, its N- and C-terminus protrude into the cytoplasm and contribute to its function. Additionally, we describe a yeast homologue of Der1p, Dfm1p, which does not seem to be involved in ERAD. In contrast, a Caenorhabditis elegans orthologue of Der1p, R151.6, is capable of complementing der1-defective phenotypes in yeast.     

A RING-H2 finger motif is essential for the function of Der3/Hrd1 in endoplasmic reticulum associated protein degradation in the yeast Saccharomyces cerevisiae

Der3/Hrd1p is a protein required for proper degradation of misfolded soluble and integral membrane proteins in the endoplasmic reticulum (ER) in the yeast Saccharomyces cerevisiae. It is located to the ER membrane and consists of a N-terminal hydrophobic region with several transmembrane domains and a large hydrophilic tail oriented to the ER lumen containing a RING finger motif of the H2 class. We had previously reported that a truncated version of Der3p, Der3deltaRp, lacking 111 residues of the lumenal domain including the RING finger motif is not functional, suggesting the involvement of this domain in the function of the protein in ER degradation. We substantiated this hypothesis by constructing a mutated form of Der3/Hrd1p replacing the last cysteine of the motif with a serine. This mutated Der3(C399S) protein maintains the correct localization and topology of the wild-type protein, however, is not able to support the degradation of soluble and integral membrane proteins. This point mutation altering the RING-H2 motif behaves as a dominant allele especially when overexpressed from a 2mu plasmid by this increasing the half-life of CPY* more than 6-fold when compared with a wild-type strain. Furthermore coexpression of der3(C399S) with the wild-type allele is also able to partially suppress the temperature sensitive growth phenotype of a sec61-2 strain. Finally we have shown that overexpression of Hrd3p suppresses the dominant effect of the der3(C399S) mutation. These results could be explained by a competition between wild-type and mutant Der3 protein for the interaction with some other component of the ER degradation pathway, probably Hrd3p.     

Endoplasmic reticulum degradation requires lumen to cytosol signaling. Transmembrane control of Hrd1p by Hrd3p

Endoplasmic reticulum (ER)-associated degradation (ERAD) is required for ubiquitin-mediated destruction of numerous proteins. ERAD occurs by processes on both sides of the ER membrane, including lumenal substrate scanning and cytosolic destruction by the proteasome. The ER resident membrane proteins Hrd1p and Hrd3p play central roles in ERAD. We show that these two proteins directly interact through the Hrd1p transmembrane domain, allowing Hrd1p stability by Hrd3p-dependent control of the Hrd1p RING-H2 domain activity. Rigorous reevaluation of Hrd1p topology demonstrated that the Hrd1p RING-H2 domain is located and functions in the cytosol. An engineered, completely lumenal, truncated version of Hrd3p functioned normally in both ERAD and Hrd1p stabilization, indicating that the lumenal domain of Hrd3p regulates the cytosolic Hrd1p RING-H2 domain by signaling through the Hrd1p transmembrane domain. Additionally, we identified a lumenal region of Hrd3p dispensable for regulation of Hrd1p stability, but absolutely required for normal ERAD. Our studies show that Hrd1p and Hrd3p form a stoichiometric complex with ERAD determinants in both the lumen and the cytosol. The HRD complex engages in lumen to cytosol communication required for regulation of Hrd1p stability and the coordination of ERAD events on both sides of the ER membrane.     

Staring,a novel E3 ubiquitin-protein ligase that targets syntaxin 1 for degradation

Syntaxin 1 is an essential component of the neurotransmitter release machinery, and regulation of syntaxin 1 expression levels is thought to contribute to the mechanism underlying learning and memory. However, the molecular events that control the degradation of syntaxin 1 remain undefined. Here we report the identification and characterization of a novel RING finger protein, Staring, that interacts with syntaxin 1. Staring is expressed throughout the brain, where it exists in both cytosolic and membrane-associated pools. Staring binds and recruits the brain-enriched E2 ubiquitin-conjugating enzyme UbcH8 to syntaxin 1 and facilitates the ubiquitination and proteasome-dependent degradation of syntaxin 1. These findings suggest that Staring is a novel E3 ubiquitin-protein ligase that targets syntaxin 1 for degradation by the ubiquitin-proteasome pathway.     

The interferon-inducible ubiquitin-protein isopeptide ligase (E3) EFP also functions as an ISG15 E3 ligase

The expression of the ubiquitin-like protein ISG15 and protein modification by ISG15 (ISGylation) are strongly activated by interferons. Accordingly, ISG15 expression and protein ISGylation are strongly activated upon viral and bacterial infections and during other stress conditions, suggesting important roles for the ISG15 system in innate immune responses. Here, we report the identification of the ubiquitin-protein isopeptide ligase (E3) EFP (estrogen-responsive finger protein) as the ISG15 E3 ligase for 14-3-3sigma protein. Like other known components of the protein ISGylation system (ISG15, UBE1L, UBP43, and UBC8), EFP is also an interferon-inducible protein. Expression of EFP small interfering RNA decreased the ISGylation of 14-3-3sigma in the 293T cell ISGylation system as well as in MCF-7 cells upon interferon treatment. Furthermore, the ISGylation enzyme activity of EFP was RING domain-dependent. These findings indicate that EFP is an ISG15 E3 ligase for 14-3-3sigma in vivo. The fact that both UBC8 and EFP are common components in the ubiquitin and ISG15 conjugation pathways suggests a mechanism whereby a limited set of enzymes accomplishes diverse post-translational modifications of their substrates in response to changes in environmental stimulations.     

Der3p/Hrd1p Is Required for Endoplasmic Reticulum-associated Degradation of Misfolded Lumenal and Integral Membrane Proteins

We have studied components of the endoplasmic reticulum (ER) proofreading and degradation system in the yeast Saccharomyces cerevisiae. Using a der3–1 mutant defective in the degradation of a mutated lumenal protein, carboxypeptidase yscY (CPY*), a gene was cloned which encodes a 64-kDa protein of the ER membrane. Der3p was found to be identical with Hrd1p, a protein identified to be necessary for degradation of HMG-CoA reductase. Der3p contains five putative transmembrane domains and a long hydrophilic C-terminal tail containing a RING-H2 finger domain which is oriented to the ER lumen. Deletion of DER3 leads to an accumulation of CPY* inside the ER due to a complete block of its degradation. In addition, a DER3 null mutant allele suppresses the temperature-dependent growth phenotype of a mutant carrying the sec61–2 allele. This is accompanied by the stabilization of the Sec61–2 mutant protein. In contrast, overproduction of Der3p is lethal in a sec61–2 strain at the permissive temperature of 25°C. A mutant Der3p lacking 114 amino acids of the lumenal tail including the RING-H2 finger domain is unable to mediate degradation of CPY* and Sec61–2p. We propose that Der3p acts prior to retrograde transport of ER membrane and lumenal proteins to the cytoplasm where they are subject to degradation via the ubiquitin-proteasome system. Interestingly, in ubc6-ubc7 double mutants, CPY* accumulates in the ER, indicating the necessity of an intact cytoplasmic proteolysis machinery for retrograde transport of CPY*. Der3p might serve as a component programming the translocon for retrograde transport of ER proteins, or it might be involved in recognition through its lumenal RING-H2 motif of proteins of the ER that are destined for degradation.     

Targeting of gp78 for ubiquitin-mediated proteasomal degradation by Hrd1: Cross-talk between E3s in the endoplasmic reticulum

There are an increasing number of ubiquitin ligases (E3s) implicated in endoplasmic reticulum (ER)-associated degradation (ERAD) in mammals. The two for which the greatest amount of information exists are the RING finger proteins gp78 and Hrd1, which are the structural orthologs of the yeast ERAD E3 Hrd1p. We now report that Hrd1, also known as synoviolin, targets gp78 for proteasomal degradation independent of the ubiquitin ligase activity of gp78, without evidence of a reciprocal effect. This degradation is observed in mouse embryonic fibroblasts lacking Hrd1, as well as with acute manipulation of Hrd1. The significance of this is underscored by the diminished level of a gp78-specific substrate, Insig-1, when Hrd1 expression is decreased and gp78 levels are consequently increased. These finding demonstrate a previously unappreciated level of complexity of the ubiquitin system in ERAD and have potentially important ramifications for processes where gp78 is implicated including regulation of lipid metabolism, metastasis, cystic fibrosis and neurodegenerative disorders.     

Membrane topology of the endoplasmic reticulum to Golgi transport factor Erv29p

Secretory proteins are transported from the endoplasmic reticulum to the Golgi apparatus via COPII-coated intermediates. Yeast Erv29p is a transmembrane protein cycling between these compartments. It is conserved across species, with one ortholog found in each genome studied, including the surf-4 protein in mammals. Yeast Erv29p acts as a receptor, loading a specific subset of soluble cargo, including glycosylated alpha factor pheromone precursor and carboxypeptidase Y, into vesicles. As the eukaryotic secretory pathway is highly conserved, mammalian surf-4 may perform a similar role in the transport of unknown substrates. Here we report the membrane topology of yeast Erv29p, which we solved by minimally invasive cysteine accessibility scanning using thiol-specific biotinylation and fluorescent labeling methods. Erv29p contains four transmembrane domains with both termini exposed to the cytosol. Two luminal loops may contain a recognition site for hydrophobic export signals on soluble cargo.     

Membrane topology of the endoplasmic reticulum to Golgi transport factor Erv29p

Secretory proteins are transported from the endoplasmic reticulum to the Golgi apparatus via COPII-coated intermediates. Yeast Erv29p is a transmembrane protein cycling between these compartments. It is conserved across species, with one ortholog found in each genome studied, including the surf-4 protein in mammals. Yeast Erv29p acts as a receptor, loading a specific subset of soluble cargo, including glycosylated alpha factor pheromone precursor and carboxypeptidase Y, into vesicles. As the eukaryotic secretory pathway is highly conserved, mammalian surf-4 may perform a similar role in the transport of unknown substrates. Here we report the membrane topology of yeast Erv29p, which we solved by minimally invasive cysteine accessibility scanning using thiol-specific biotinylation and fluorescent labeling methods. Erv29p contains four transmembrane domains with both termini exposed to the cytosol. Two luminal loops may contain a recognition site for hydrophobic export signals on soluble cargo.     

Drosophila melanogaster Parkin ubiquitinates peanut and septin1 as an E3 ubiquitin-protein ligase

Autosomal recessive juvenile parkinsonism (AR-JP), a common familial form of Parkinson's disease, is caused by mutations of human Parkin. To deepen the understanding of Parkin biology in an in vivo model of Drosophila, we attempted to characterize the function of Drosophila melanogaster Parkin and found that D. melanogaster Parkin exhibited UbcH8-dependent E3 ubiquitin-protein ligase activity. Using E2 binding and in vitro ubiquitination assays, UbcH8 preferentially was found to bind to Parkin mutants harboring functional RING1 domains, but failed to bind to mutants harboring point mutants with complete loss of function. This inability of UbcH8 binding to such mutants was accompanied by abrogation of an E3 ligase activity, indicating that D. melanogaster Parkin as an E3 ligase interacts with UbcH8 through its RING1 domain. An in vivo ubiquitination assay revealed that D. melanogaster Parkin existed in ubiquitinated form in vivo. Moreover, peanut and septin1, D. melanogaster septin proteins, were also ubiquitinated by D. melanogaster Parkin. Co-immunoprecipitation with membrane protein Syntaxin indicated direct binding of septin proteins to syntaxin, implicating their relevance in the exocytosis of dopamine in cells. Western blot analysis and DNA fragmentation indicated that the rate and efficiency of p53-dependent apoptosis were significantly higher in the presence of dopamine than without the septin proteins. Therefore, our findings in the present study demonstrate that Parkin possibly influences septin protein effects on p53-mediated apoptosis, helping to extend the utility of Drosophila as a model system for the study of neurodegeneration.     

Ubiquitin ligase Kf-1 is involved in the endoplasmic reticulum-associated degradation pathway

Kf-1 was first identified as a gene showing enhanced expression in the cerebral cortex of a sporadic Alzheimer’s disease patient. To date, however, the functional properties of Kf-1 protein remain unknown. In this study, immunohistochemical analysis showed that Kf-1 immunoreactivity was detected in rat hippocampus and cerebral cortex neurons. Interestingly, it was colocalized with endoplasmic reticulum (ER) marker. To investigate the specific function of Kf-1 protein, we generated Myc tagged wild type Kf-1 (Myc-Kf-1WT) and RING finger domain deletion mutant of Kf-1 (Myc-Kf-1ΔR), and then transfected in HEK293 cells. Myc-Kf-1WT displayed a reticular pattern typical of ER localization, with large perinuclear aggregates and colocalized with ER marker, calnexin. Myc-Kf-1WT facilitated ubiquitination of endogenous proteins, whereas Myc-Kf-1ΔR did not show ubiquitin ligase activity. In addition, we found that Kf-1 interacted with components of the ER-associated degradation (ERAD) pathway, including Derlin-1 and VCP. Taken together, these properties suggest that Kf-1 is an ER ubiquitin ligase involved in the ERAD pathway.     

The E3 ligase Smurf1 regulates Wolfram syndrome protein stability at the endoplasmic reticulum

The HECT-type ubiquitin ligase (E3) Smad ubiquitination regulatory factor 1 (Smurf1) targets various substrates, including Smad1/5, RhoA, Prickle 1, MEKK2, and JunB for degradation and thereby regulates adult bone formation and embryonic development. Here, we identify the endoplasmic reticulum (ER)-localized Wolfram syndrome protein (WFS1) as a specific degradation substrate of Smurf1. Mutations in the WFS1 gene cause Wolfram syndrome, an autosomal recessive disorder characterized by diabetes mellitus and optic atrophy. WFS1 negatively regulates the ER stress response, and WFS1 deficiency in mice increases ER stress and triggers apoptosis. We show that Smurf1 interacts with WFS1 at the ER and promotes the ubiquitination and proteasomal degradation of WFS1. A C-terminal luminal region in WFS1, including residues 667-700, is involved in this degradation. Wild-type WFS1 as well as a subset of WFS1 mutants that include this degron region are susceptible to Smurf1-mediated degradation. By contrast, pathophysiological deletion mutants of WFS1 lacking the degron, such as W648X, Y660X, and Q667X, are resistant to degradation by Smurf1. Depletion of Smurf1 by RNA interference results in increased WFS1 and decreased ATF6α levels. Furthermore, we show that ER stress induces Smurf1 degradation and WFS1 up-regulation. These findings reveal for the first time that Smurf1 targets an ER-localized protein for degradation and that Smurf1 is regulated by ER stress.     

A HECT E3 ubiquitin-protein ligase with sequence similarity to E6AP does not target p53 for degradation in the softshell clam (Mya arenaria)

Numerous reports have raised the level of national concern that chemicals found in the environment may have adverse effects on the health of humans and wildlife. Environmental exposure to pollutants, such as dioxin, has been implicated in gonadal tumor formation in Maine softshell clams (Mya arenaria). Prevalence of these tumors is as high as 40% in some populations. Although their etiology is still unknown, investigations into the mechanisms of tumor formation have revolved around a hypothesis of dioxin-induced toxicity. The aryl hydrocarbon receptor (AHR) was initially investigated, but was later determined to not bind the prototypical ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), suggesting that dioxin toxicity is mediated through an AHR-independent pathway. An alternative mechanism of tumor formation has been investigated, involving a protein with significant sequence similarity to mammalian E6AP, a HECT (homologous to E6AP carboxy terminus) E3 ubiquitin-protein ligase. E6AP, in association with the high-risk human papillomavirus (HPV) E6 protein, is involved in the abnormal degradation of the p53 tumor suppressor protein in human cervical cancer. Tumorigenic clam reproductive tissue revealed higher M. arenaria E3 (MaE3) protein levels concomitant with lower M. arenaria p53 (Map53) levels. While the function of MaE3 as a HECT E3 was verified, results from three methods agree that MaE3 does not associate with Map53. However, alteration in Map53 levels may still play a role in clam gonadal tumorigenesis. Due to upregulation of MaE3 in neoplastic reproductive tissue, further investigations will focus on determining the proteolytic targets of MaE3. In conjunction with our previous findings that dioxin toxicity in the softshell clam is not mediated by AHR, the results from our current investigation suggest a complex etiology for the clam germinomas.     

Rtn1p is involved in structuring the cortical endoplasmic reticulum

The endoplasmic reticulum (ER) contains both cisternal and reticular elements in one contiguous structure. We identified rtn1Delta in a systematic screen for yeast mutants with altered ER morphology. The ER in rtn1Delta cells is predominantly cisternal rather than reticular, yet the net surface area of ER is not significantly changed. Rtn1-green fluorescent protein (GFP) associates with the reticular ER at the cell cortex and with the tubules that connect the cortical ER to the nuclear envelope, but not with the nuclear envelope itself. Rtn1p overexpression also results in an altered ER structure. Rtn proteins are found on the ER in a wide range of eukaryotes and are defined by two membrane-spanning domains flanking a conserved hydrophilic loop. Our results suggest that Rtn proteins may direct the formation of reticulated ER. We independently identified Rtn1p in a proteomic screen for proteins associated with the exocyst vesicle tethering complex. The conserved hydophilic loop of Rtn1p binds to the exocyst subunit Sec6p. Overexpression of this loop results in a modest accumulation of secretory vesicles, suggesting impaired exocyst function. The interaction of Rtn1p with the exocyst at the bud tip may trigger the formation of a cortical ER network in yeast buds.     

Der1, a novel protein specifically required for endoplasmic reticulum degradation in yeast.

The endoplasmic reticulum (ER) of the yeast Saccharomyces cerevisiae contains of proteolytic system able to selectively degrade misfolded lumenal secretory proteins. For examination of the components involved in this degradation process, mutants were isolated. They could be divided into four complementation groups. The mutations led to stabilization of two different substrates for this process. The mutant classes were called ''der'' for ''degradation in the ER''. DER1 was cloned by complementation of the der1-2 mutation. The DER1 gene codes for a novel, hydrophobic protein, that is localized to the ER. Deletion of DER1 abolished degradation of the substrate proteins. The function of the Der1 protein seems to be specifically required for the degradation process associated with the ER. The depletion of Der1 from cells causes neither detectable growth phenotypes nor a general accumulation of unfolded proteins in the ER. In DER1-deleted cells, a substrate protein for ER degradation is retained in the ER by the same mechanism which also retains lumenal ER residents. This suggests that DER1 acts in a process that directly removes protein from the folding environment of the ER.     

Cytosolic entry of Shiga-like toxin a chain from the yeast endoplasmic reticulum requires catalytically active Hrd1p

Background

Escherichia coli Shiga-like toxin 1 normally traffics to the endoplasmic reticulum (ER) in sensitive mammalian cells from where the catalytic A chain (SLTxA1) dislocates to the cytosol to inactivate ribosomes. Currently, no molecular details of the dislocation process are available. To investigate the mechanism of the dislocation step we expressed SLTxA1 in the ER of Saccharomyces cerevisiae.

Methodology and Principal Findings

Using a combination of growth studies and biochemical tracking in yeast knock-out strains we show that SLTxA1 follows an ER-associated degradation (ERAD) pathway to enter the cytosol in a step mediated by the transmembrane Hrd1p ubiquitin ligase complex. ER-to-cytosol dislocation of the bulk population of SLTxA1 requires Cdc48p and its ubiquitin-handling co-factor Npl4p, and this population of toxin is terminally dispatched by proteasomal degradation. A small sub-population of SLTxA1 uncouples from this classical ERAD pathway and recovers catalytic activity in the cytosol. The pathway that leads to toxicity is also Hrd1p-dependent but, unlike that for the related ricin A chain toxin, SLTxA1 dislocation does require the catalytic cysteine of Hrd1p. However it does not depend on canonical ubiquitylation since toxin variants lacking endogenous lysyl residues also utilize this pathway, and furthermore there is no requirement for a number of Cdc48p co-factors.

Conclusions and Significance

The fraction of SLTxA1 that disengages from the ERAD pathway thus does so upstream of Cdc48p interactions and downstream of Hrd1p interactions, in a step that possibly involves de-ubiquitylation. Mechanistically therefore, the dislocation of this toxin is quite distinct from that of conventional ERAD substrates that are normally degraded, and the toxins partially characterised to date that do not require the catalytic cysteine of the major Hrd1p component of the dislocation apparatus.     

Bifunctional apoptosis regulator (BAR), an endoplasmic reticulum (ER)-associated E3 ubiquitin ligase, modulates BI-1 protein stability and function in ER Stress

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates inositol-requiring protein-1 (IRE1), among other ER-associated signaling proteins of the unfolded protein response (UPR) in mammalian cells. IRE1 signaling becomes attenuated under prolonged ER stress. The mechanisms by which this occurs are not well understood. An ER resident protein, Bax inhibitor-1 (BI-1), interacts with IRE1 and directly inhibits IRE1 activity. However, little is known about regulation of the BI-1 protein. We show here that bifunctional apoptosis regulator (BAR) functions as an ER-associated RING-type E3 ligase, interacts with BI-1, and promotes proteasomal degradation of BI-1. Overexpression of BAR reduced BI-1 protein levels in a RING-dependent manner. Conversely, knockdown of endogenous BAR increased BI-1 protein levels and enhanced inhibition of IRE1 signaling during ER stress. We also found that the levels of endogenous BAR were reduced under prolonged ER stress. Our findings suggest that post-translational regulation of the BI-1 protein by E3 ligase BAR contributes to the dynamic control of IRE1 signaling during ER stress.     

RING finger palmitoylation of the endoplasmic reticulum Gp78 E3 ubiquitin ligase

Gp78 is an E3 ubiquitin ligase within the endoplasmic reticulum-associated degradation pathway. We show that Flag-tagged gp78 undergoes sulfhydryl cysteine palmitoylation (S-palmitoylation) within the RING finger motif, responsible for its ubiquitin ligase activity. Screening of 19 palmitoyl acyl transferases (PATs) identified five that increased gp78 RING finger palmitoylation. Endoplasmic reticulum (ER)-localized Myc-DHHC6 overexpression promoted the peripheral ER distribution of Flag-gp78 while RING finger mutation and the palmitoylation inhibitor 2-bromopalmitate restricted gp78 to the central ER. Palmitoylation of RING finger cysteines therefore regulates gp78 distribution to the peripheral ER.