Peripheral ER Research Articles

8595 Brain Penetration Impedes SERD Efficacy in Breast Cancer Pre-clinical Models

Abstract Disclosure: S.E. Wardell: None. B. Chakraborty: None. S. Artham: None. M.A. Newlin: None. M. Kirkland: None. C. Chang: None. D.P. McDonnell: None. Most breast cancers express the estrogen receptor (ER, ESR1), and agents that target ER remain the cornerstone of interventions used to treat this disease at all stages. Aromatase inhibitors (AI), which block estrogen synthesis, and the selective ER downregulator (SERD) fulvestrant were developed with the goal of achieving absolute inhibition of ER signaling in tumors. However, it has been assumed that the clinical efficacy (and utility) of fulvestrant is somewhat limited by its poor pharmaceutical properties and the requirement that it be administered by intramuscular injection. This, together with the observation that the activating ESR1 mutations selected for during AI treatment are not effectively inhibited by fulvestrant, encouraged the development of new classes of SERDs that achieve sufficient exposure to saturate and degrade ER in tumors. The recent approval of the second-generation SERD elacestrant for the treatment of breast cancers harboring ESR1 mutations was a major advance in approaches to target ER in advanced breast cancer. Several additional oral SERDs have been developed and evaluated as potential treatments for advanced metastatic breast cancer. Despite showing substantial efficacy in pre-clinical assays that assess ER downregulation and gene transcription, and notably equivalent antitumor activities in cell line derived and PDX tumors in immunocompromised mice, the clinical development of the majority of these SERDs has been discontinued for lack of efficacy. In reviewing the available clinical data across many SERD programs, we noted that some drugs demonstrated a paradoxical, non-linear response curve with higher doses exhibiting less efficacy than lower doses. Probing this pharmacology in animal models we determined that the ability of SERDs to cross the blood brain barrier and attenuate ER signaling in the hypothalamus was a key differentiator of SERDs. In validated syngeneic models of breast cancer, we observed doses of fulvestrant having confirmed brain exposure stimulated tumor growth independent of tumor ER-status. We evaluated the ability of several next generation SERDs for their brain penetrance and demonstrated that SERD presence in the brain was associated with a stimulatory effect on tumor growth. Interestingly, administration of a low dose of 17b-estradiol (E2) together with a brain excluded SERD further repressed tumor growth, highlighting a potentially beneficial anti-tumor role for estrogen signaling in the brain. Furthermore, combination of a brain excluded SERM with E2 also resulted in increased tumor growth repression; thus, ER downregulation is dispensable for the cooperative activity observed for estrogens administered together with a peripheral ER antagonist. These data suggest that differences in tissue pharmacology, as opposed to differences in ability to degrade ER, is likely to be a major differentiator of ER modulators. Presentation: 6/3/2024

Preparation of Highly Enriched ER Membranes Using Free-Flow Electrophoresis.

Free-flow electrophoresis (FFE) is a technique for separation of proteins, peptides, organelles, and cells. With zone electrophoresis (ZE-FFE), organelles are separated according to surface charge. The ER is the only remaining major cellular compartment in Arabidopsis not to have been isolated using density centrifugation, immune-isolation, or any other method previously applied to purification of plant membranes. By using continuous-flow electrophoresis, ER vesicles of similar surface charge, which may have been fragmented during cell lysis, can be focused. A large portion of these vesicles are of sufficiently different surface charge that separation from the majority of Golgi and other contaminants is possible. Here we adapt an earlier ZE-FFE Golgi isolation protocol for the isolation of highly pure ER vesicles and for tracking the migration of peripheral ER vesicles. Isolating ER vesicles of homogeneous surface charge allows multi-omic analyses to be performed on the ER. This facilitates investigations into structure-function relationships within the ER.

Comparative membrane proteomics reveals diverse cell regulators concentrated at the nuclear envelope.

The nuclear envelope (NE) is a subdomain of the ER with prominent roles in nuclear organization, which are largely mediated by its distinctive protein composition. We developed methods to reveal low-abundance transmembrane (TM) proteins concentrated at the NE relative to the peripheral ER. Using label-free proteomics that compared isolated NEs with cytoplasmic membranes, we first identified proteins with apparent NE enrichment. In subsequent authentication, ectopically expressed candidates were analyzed by immunofluorescence microscopy to quantify their targeting to the NE in cultured cells. Ten proteins from a validation set were found to associate preferentially with the NE, including oxidoreductases, enzymes for lipid biosynthesis, and regulators of cell growth and survival. We determined that one of the validated candidates, the palmitoyltransferase Zdhhc6, modifies the NE oxidoreductase Tmx4 and thereby modulates its NE levels. This provides a functional rationale for the NE concentration of Zdhhc6. Overall, our methodology has revealed a group of previously unrecognized proteins concentrated at the NE and additional candidates. Future analysis of these can potentially unveil new mechanistic pathways associated with the NE.

Dual control of formin-nucleated actin assembly by the chromatin and ER in mouse oocytes

The first asymmetric meiotic cell divisions in mouse oocytes are driven by formin 2 (FMN2)-nucleated actin polymerization around the spindle. In this study, we investigated how FMN2 is recruited to the spindle peripheral ER and how its activity is regulated in mouse meiosis I (MI) oocytes. We show that this process is regulated by the Ran GTPase, a conserved mediator of chromatin signal, and the ER-associated protein VAPA. FMN2 contains a nuclear localization sequence (NLS) within a domain (SLD) previously shown to be required for FMN2 localization to the spindle periphery. FMN2 NLS is bound to the importin α1/β complex, and the disruption of this interaction by RanGTP is required for FMN2 accumulation in the area proximal to the chromatin and the MI spindle. The importin-free FMN2 is then recruited to the surface of ER around the spindle through the binding of the SLD with the ER-membrane protein VAPA. We further show that FMN2 is autoinhibited through an intramolecular interaction between the SLD with the C-terminal formin homology 2 (FH2) domain that nucleates actin filaments. VAPA binding to SLD relieves the autoinhibition of FMN2, leading to localized actin polymerization. This dual control of formin-mediated actin assembly allows actin polymerization to initiate the movement of the meiotic spindle toward the cortex, an essential step in the maturation of the mammalian female gamete.

LIPID transfer proteins regulate store-operated calcium entry via control of plasma membrane phosphoinositides

The ER-resident proteins STIM1 together with the plasma membrane (PM)-localized Orai1 channels constitute the molecular components of the store-operated Ca2+ entry (SOCE) pathway. Prepositioning of STIM1 to the peripheral ER close to the PM ensures its efficient interaction with Orai1 upon a decrease in the ER luminal Ca2+ concentration. The C-terminal polybasic domain of STIM1 has been identified as mediating the interaction with PM phosphoinositides and hence positions the molecule to ER-PM contact sites. Here we show that STIM1 requires PM phosphatidylinositol 4-phosphate (PI4P) for efficient PM interaction. Accordingly, oxysterol binding protein related proteins (ORPs) that work at ER-PM junctions and consume PI4P gradients exert important control over the Ca2+ entry process. These studies reveal an important connection between non-vesicular lipid transport at ER-PM contact sites and regulation of ER Ca2+store refilling.

CDK1 controls CHMP7-dependent nuclear envelope reformation.

Through membrane sealing and disassembly of spindle microtubules, the Endosomal Sorting Complex Required for Transport-III (ESCRT-III) machinery has emerged as a key player in the regeneration of a sealed nuclear envelope (NE) during mitotic exit, and in the repair of this organelle during interphase rupture. ESCRT-III assembly at the NE occurs transiently during mitotic (M) exit and is initiated when CHMP7, an ER-localised ESCRT-II/ESCRT-III hybrid protein, interacts with the Inner Nuclear Membrane (INM) protein LEM2. Whilst classical nucleocytoplasmic transport mechanisms have been proposed to separate LEM2 and CHMP7 during interphase, it is unclear how CHMP7 assembly is suppressed in mitosis when NE and ER identities are mixed. Here, we use live cell imaging and protein biochemistry to examine the biology of these proteins during M-exit. Firstly, we show that CHMP7 plays an important role in the dissolution of LEM2 clusters that form at the NE during M-exit. Secondly, we show that CDK1 phosphorylates CHMP7 upon M-entry at Ser3 and Ser441 and that this phosphorylation reduces CHMP7's interaction with LEM2, limiting its assembly during M-phase. We show that spatiotemporal differences in the dephosphorylation of CHMP7 license its assembly at the NE during telophase, but restrict its assembly on the ER at this time. Without CDK1 phosphorylation, CHMP7 undergoes inappropriate assembly in the peripheral ER during M-exit, capturing LEM2 and downstream ESCRT-III components. Lastly, we establish that a microtubule network is dispensable for ESCRT-III assembly at the reforming nuclear envelope. These data identify a key cell-cycle control programme allowing ESCRT-III-dependent nuclear regeneration.

Fine-tuning ER-phagy by post-translational modifications.

Autophagy functions in both selective and non-selective ways to maintain cellular homeostasis. Endoplasmic reticulum autophagy (ER-phagy) is a subclass of autophagy responsible for the degradation of the endoplasmic reticulum through selective encapsulation into autophagosomes. ER-phagy occurs both under physiological conditions and in response to stress cues, and plays a crucial role in maintaining the homeostatic control of the organelle. Although specific receptors that target parts of the ER membrane, as well as, internal proteins for lysosomal degradation have been identified, the molecular regulation of ER-phagy has been elusive. Recent work has uncovered novel regulators of ER-phagy that involve post-translational modifications of ER-resident proteins and functional cross-talk with other cellular processes. Herein, we discuss how morphology affects the function of the peripheral ER, and how ER-phagy modulates the turnover of this organelle. We also address how ER-phagy is regulated at the molecular level, considering implications relevant to human diseases.

The ORMs interact with transmembrane domain 1 of Lcb1 and regulate serine palmitoyltransferase oligomerization, activity and localization

Serine palmitoyltransferase (SPT), an endoplasmic reticulum-localized membrane enzymecomposed of acatalytic LCB1/LCB2 heterodimer and a small activating subunit (Tsc3 in yeast; ssSPTs in mammals), is negatively regulated by the evolutionarily conserved family of proteins known as the ORMs. In yeast, SPT, the ORMs, and the PI4P phosphatase Sac1, copurify in the “SPOTs” complex. However, neither the mechanism of ORM inhibition of SPT nor details of the interactions of the ORMs and Sac1 with SPT are known. Here we report that the first transmembrane domain (TMD1) of Lcb1 is required for ORM binding to SPT. Loss of binding is not due to altered membrane topology of Lcb1 since replacing TMD1 with a heterologous TMD restores membrane topology but not ORM binding. TMD1 deletion also eliminates ORM-dependent formation of SPT oligomers as assessed by co-immunoprecipitation assays and in vivo imaging. Expression of ORMs lacking derepressive phosphorylation sites results in constitutive SPT oligomerization, while phosphomimetic ORMs fail to induce oligomerization under any conditions. Significantly, when LCB1–RFP and LCB1ΔTMD1–GFP were coexpressed, more LCB1ΔTMD1–GFP was in the peripheral ER, suggesting ORM regulation is partially accomplished by SPT redistribution. Tsc3 deletion does not abolish ORM inhibition of SPT, indicating the ORMs do not simply prevent activation by Tsc3. Binding of Sac1 to SPT requires Tsc3, but not the ORMs, and Sac1 does not influence ORM-mediated oligomerization of SPT. Finally, yeast mutants lacking ORM regulation of SPT require the LCB-P lyase Dpl1 to maintain long-chain bases at sublethal levels.

Calcium and lipid exchange machinery in Sertoli cells

Sperm production (spermatogenesis) takes place in the seminiferous tubules of the testis. Specialized junction complexes termed ‘ectoplasmic specializations' (ESs) in the seminiferous epithelium have several key functions. ESs between Sertoli cells disassemble during the movement of early spermatogenic cells towards the apex of the epithelium and those ESs between a Sertoli cell and late stage spermatids disassemble during sperm release. There is evidence that structures called ‘Tubulobulbar Complexes' (TBCs) are endocytic structures responsible for the internalization and eventual degradation of junctions associated with ESs, allowing for early spermatocyte translocation from the basal epithelium and release of late spermatids from the apical epithelium. One key feature of both ESs and TBCs is their extensive association with peripheral ER. ESs are tripartite in structure consisting of Sertoli cell plasma membrane (PM), a layer of actin bundles, and an overlying leaflet of ER. TBCs are double membrane invaginations that appear as ESs are beginning to break down. They are long, endocytic in‐growths, with a swelled bulb region that contains an extensive ER‐PM contact site. TBC bulbs eventually ‘bud’, fuse, and become endosomes. Little is understood about the function of the ER at both ESs and TBCs, though it is well established that ER contact sites in other cells function in both calcium signalling and lipid exchange. If ER in ESs and ER‐TBC contacts function in calcium and lipid exchange, then known calcium signalling machinery and lipid exchange proteins should be localized to these structures. Using confocal imaging, we localized the CACNA1H voltage gated calcium channel to the PM of apical ESs, and the VAPA integral ER protein, which is associated with lipid synthesis and exchange machinery, to the apical ER as well as the ER in basal regions known to contain ESs. This data adds further weight to the hypothesis that calcium exchange is integral to regulation of ES and TBC structure, as well as begins our exploration into the possible role of lipid exchange at these sites.Support or Funding InformationThis work was supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada (155397‐2013 to A. Wayne Vogl)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Uncovering Theorganelle Interactome: Dynamic Imaging of Multiple Organelles

Numerous aspects of cell biology and biophysics are becoming clearer due to emerging visualization technologies, which can capture processes at the level of whole organisms down to single molecules. Here, I will discuss developments in probes and microscopes that are dramatically expanding productive imaging. To surmount fluorophore bleed-through, a combined excitation-based spectral unmixing and lattice light sheet microscopy was used to visualize up to six organelles (i.e., ER, Golgi, mitochondria, lysosomes, peroxisomes and lipid droplets) simultaneously within cells. This allowed us to track these organelles through time and analyze their inter-organelle contacts, providing a systems-level map of the organelle interactome and how it is perturbed under different physiological conditions. To increase temporal resolution during imaging, we employed total internal reflection fluorescence combined with structured illumination microscopy to visualize organelle dynamics at very high temporal-spatial resolution. Examining the ER, we observed that many peripheral ER sheets seen using diffraction-limited imaging are actually highly perforated structures comprised of tightly latticed groups of dynamic tubules. Within the latticed ER tubule meshwork, subdiffraction-limited holes were observed (∼150-250 nm diameter) having transient lifespans (∼250 msec). Viewed at higher resolution using lattice light sheet microscopy combined with point accumulation for nanoscale topology (PAINT), the peripheral ER sheets represented a complex meshwork of tightly cross-linked ER tubules, with potentially important roles in ER function.

Preparation of Highly Enriched ER Membranes Using Free-Flow Electrophoresis.

Free-flow electrophoresis (FFE) is a technique for separation of proteins, peptides, organelles, and cells. With zone electrophoresis (ZE-FFE), organelles are separated according to surface charge. The ER is the only remaining major cellular compartment in Arabidopsis not to have been isolated using density centrifugation, immune-isolation, or any other method previously applied to purification of plant membranes. By using continuous-flow electrophoresis ER vesicles of similar surface charge, which may have been fragmented during cell lysis, can be focused. A large portion of these vesicles are of sufficiently different surface charge that separation from the majority of Golgi and other contaminants is possible. Here we adapt an earlier ZE-FFE Golgi isolation protocol for the isolation of highly pure ER vesicles and for tracking the migration of peripheral ER vesicles. Isolating ER vesicles of homogenous surface charge allows multi-'omic analyses to be performed on the ER. This facilitates investigations into structure-function relationships within the ER.

Tracking Diacylglycerol Pools in Budding Yeast

Many diacylglycerol (DAG) sensing probes have been developed to monitor DAG signalling in mammalian cells. Several C1 domains have been fused to fluorescent proteins that can sense DAG in cellular membranes. The C1 domain of PKCδ is very well characterized and known to bind DAG with high affinity, and has been successfully used as a probe fused to GFP in order to monitor DAG in live cells. We have cloned the C1 domain of PKCδ (C1δ) into a centromeric yeast expression vector under a constitutive promoter for in vivo visualization of DAG pools. Localization of C1δ‐GFP was first monitored in different wild type strains grown under standard conditions. The main localization of this DAG‐binding reporter in actively growing cells was the vacuolar membrane, further confirmed by co‐localization with FM4‐64. This localization is consistent with studies done using similar probes to show that DAG localizes to the membrane of isolated vacuoles from yeast cells. Importantly, no changes in vacuolar morphology of wild type cells due to expression of C1δ‐GFP were detected. In addition to vacuolar membrane localization, the C1δ probe also associated with the plasma membrane of daughter cells and at the bud neck. In contrast to the phosphatidic acid (PA)‐binding reporter GFP‐Spo2051–91, C1δ‐GFP did not show nuclear localization as determined by co‐localization analysis using the nucleolar marker Sik1‐RFP. We then tested if the localization would change in conditions where lipid homeostasis and levels of DAG are known to be altered. For this, we used cells lacking the four enzymes responsible for the final step in the synthesis of triglycerides in yeast, namely DAG acyltransferase Dga1, the transacylase Lro1, and the steryl acyltransferases Are1 and Are2 (4Δ quadruple mutant), which accumulate DAG and have defects in both nuclear and peripheral ER membrane organization. Localization of the C1δ‐GFP probe drastically changed when expressed in 4Δ quadruple mutant cells, clearly revealing its association with the expanded nuclear and cortical ER in addition to the vacuolar membrane. Additionally, the polarized distribution of C1δ‐GFP to the plasma membrane of buds and daughter cells significantly decreased in the quadruple mutant. After validating the use of this probe in yeast, we investigated the localization of DAG pools in response to lysophosphatidylcholine (LysoPC) analogues, as we have determined they induce a 5 times increase in DAG levels. Interestingly, incubation with LysoPC analogues induced a drastic change in the localization of DAG while no differences were observed regarding the distribution of PA. These results further support the use of C1δ‐GFP as a valuable tool to track cytosolic accessible pools of DAG in yeast.Support or Funding InformationThis work was supported by an operating grant from the Natural Sciences and Engineering Research Council of Canada to VZ

Cell biology and the "real world".

Cell biology and the "real world".

PM365 Effects of Androgen Replacement on Age-Related Impairment in Ischaemia-Mediated Neovascularisation

O ST E R A B ST R A cardiac, peripheral and pulmonary ER has been quantified to assess BNP synthesis and clearance. We assumed that the pulmonary ER would represent NEP depending clearance pathway. Results: The mean serum BNP level was 37ng/L. The mean cardiac ER was 1.20, pulmonary and peripheral ER was 0.13. There was a tendency for lower cardiac and pulmonary ER in males. Pulmonary ER correlated with age (r1⁄40.495, p1⁄40.023) and systolic BP (r1⁄40.58, p1⁄40.006) only. However there was no correlation between cardiac and pulmonary ER with BMI, waist circumference and HOMA-IR.

A spastic paraplegia mouse model reveals REEP1-dependent ER shaping

Axonopathies are a group of clinically diverse disorders characterized by the progressive degeneration of the axons of specific neurons. In hereditary spastic paraplegia (HSP), the axons of cortical motor neurons degenerate and cause a spastic movement disorder. HSP is linked to mutations in several loci known collectively as the spastic paraplegia genes (SPGs). We identified a heterozygous receptor accessory protein 1 (REEP1) exon 2 deletion in a patient suffering from the autosomal dominantly inherited HSP variant SPG31. We generated the corresponding mouse model to study the underlying cellular pathology. Mice with heterozygous deletion of exon 2 in Reep1 displayed a gait disorder closely resembling SPG31 in humans. Homozygous exon 2 deletion resulted in the complete loss of REEP1 and a more severe phenotype with earlier onset. At the molecular level, we demonstrated that REEP1 is a neuron-specific, membrane-binding, and membrane curvature-inducing protein that resides in the ER. We further show that Reep1 expression was prominent in cortical motor neurons. In REEP1-deficient mice, these neurons showed reduced complexity of the peripheral ER upon ultrastructural analysis. Our study connects proper neuronal ER architecture to long-term axon survival.

The Aspergillus nidulans Peripheral ER: Disorganization by ER Stress and Persistence during Mitosis

The genetically amenable fungus Aspergillus nidulans is well suited for cell biology studies involving the secretory pathway and its relationship with hyphal tip growth by apical extension. We exploited live-cell epifluorescence microscopy of the ER labeled with the translocon component Sec63, endogenously tagged with GFP, to study the organization of ‘secretory’ ER domains. The Sec63 A. nidulans ER network includes brightly fluorescent peripheral strands and more faintly labeled nuclear envelopes. In hyphae, the most abundant peripheral ER structures correspond to plasma membrane-associated strands that are polarized, but do not invade the hyphal tip dome, at least in part because the subapical collar of endocytic actin patches constrict the cortical strands in this region. Thus the subapical endocytic ring might provide an attachment for ER strands, thereby ensuring that the growing tip remains ‘loaded’ with secretory ER. Acute disruption of secretory ER function by reductive stress-mediated induction of the unfolded protein response results in the reversible aggregation of ER strands, cessation of exocytosis and swelling of the hyphal tips. The secretory ER is insensitive to brefeldin A treatment and does not undergo changes during mitosis, in agreement with the reports that apical extension continues at normal rates during this period.

Lead aprons: how protected are you?

Lead aprons: how protected are you?

Self‐Organization of Transitional ER Sites

We are studying the biogenesis and dynamics of transitional ER (tER) sites using the budding yeast Pichia pastoris. Previously, we showed that P. pastoris tER sites are long‐lived structures that form de novo, fuse upon collision, and grow or shrink to attain a steady‐state size. This behavior can be explained by a self‐organization model. Specifically, we postulate that capture of new tER components is balanced by shrinkage driven by the budding of COPII coated transport vesicles. To test this model, we used a genetic screen to identify Sec16 as a key player in tER organization. Sec16 is a large peripheral ER membrane protein that interacts with multiple COPII components. Our results suggest that Sec16 acts as a negative regulator of ER export. According to this view, Sec16 functions primarily to control tER dynamics rather than to nucleate tER site formation. Sec16 defines a new level of regulation for the ER export system.

Infection-Associated Nuclear Degeneration in the Rice Blast Fungus Magnaporthe oryzae Requires Non-Selective Macro-Autophagy

BackgroundThe rice blast fungus Magnaporthe oryzae elaborates a specialized infection structure called an appressorium to breach the rice leaf surface and gain access to plant tissue. Appressorium development is controlled by cell cycle progression, and a single round of nuclear division occurs prior to appressorium formation. Mitosis is always followed by programmed cell death of the spore from which the appressorium develops. Nuclear degeneration in the spore is known to be essential for plant infection, but the precise mechanism by which it occurs is not known.Methodology/Principal FindingsIn yeast, nuclear breakdown requires a specific form of autophagy, known as piecemeal microautophagy of the nucleus (PMN), and we therefore investigated whether this process occurs in the rice blast fungus. Here, we report that M. oryzae possesses two conserved components of a putative PMN pathway, MoVac8 and MoTsc13, but that both are dispensable for nuclear breakdown during plant infection. MoVAC8 encodes a vacuolar membrane protein and MoTSC13 a peri-nuclear and peripheral ER protein.Conclusions/SignificanceWe show that MoVAC8 is necessary for caffeine resistance, but dispensable for pathogenicity of M. oryzae, while MoTSC13 is involved in cell wall stress responses and is an important virulence determinant. By functional analysis of ΔMoatg1 and ΔMoatg4 mutants, we demonstrate that infection-associated nuclear degeneration in M. oryzae instead occurs by non-selective macroautophagy, which is necessary for rice blast disease.

Peripheral Endoplasmic Reticulum Localization of Gp78 Ubiquitin Ligase Activity

Gp78 (also known as AMFR and RNF45) is an E3 ubiquitin ligase that targets proteins for proteasomal degradation through endoplasmic reticulum (ER)-associated degradation (ERAD). In this study, we showed that gp78-mediated ubiquitylation is initiated in the peripheral ER. Substrate monoubiquitylation and gp78 CUE domain integrity restricted substrate to the peripheral ER, where CUE domain interactions and polyubiquitylation reduced gp78 mobility. Derlin-1 and derlin-2, which are involved in the retrotranslocation of ERAD substrates, localized to a central, juxtanuclear ER domain, where polyubiquitylated proteins accumulated upon proteasome inhibition. Transfer of polyubiquitylated substrate to the central ER was dependent on ubiquitin chain elongation and recruitment of the AAA ATPase p97 (also known as VCP). HT-1080 fibrosarcoma cells expressed elevated levels of endogenous gp78, which was associated with segregation of ubiquitylated substrate to the peripheral ER and its polyubiquitin-dependent redistribution to the central ER upon proteasome inhibition. Therefore, the peripheral ER is the site of gp78 ubiquitin ligase activity. Delivery of ubiquitylated substrate to the central ER was regulated by ubiquitin chain elongation and opposing actions of gp78 CUE domain interactions and p97 recruitment.