J-domain proteins: From molecular mechanisms to diseases.

J-domain proteins (JDPs) are the largest family of chaperones in most organisms, but much of how they function within the network of other chaperones and protein quality control machineries is still an enigma. Here, we report on the latest findings related to JDP functions presented at a dedicated JDP workshop in Gdansk, Poland. The report does not include all (details) of what was shared and discussed at the meeting, because some of these original data have not yet been accepted for publication elsewhere or represented still preliminary observations at the time.

Novel functions of the ER-located Hsp40s DNAJB12 and DNAJB14 on proteins at the outer mitochondrial membrane under stress mediated by CCCP.

The endoplasmic reticulum (ER) membrane provides infrastructure for intracellular signaling, protein degradation, and communication among the ER lumen, cytosol, and nucleus via transmembrane and membrane-associated proteins. Failure to maintain homeostasis at the ER leads to deleterious conditions in humans, such as protein misfolding-related diseases and neurodegeneration. The ER transmembrane heat shock protein 40 (Hsp40) proteins, including DNAJB12 (JB12) and DNAJB14 (JB14), have been studied for their importance in multiple aspects of cellular events, including degradation of misfolded membrane proteins, proteasome-mediated control of proapoptotic Bcl-2 members, and assembly of multimeric ion channels. This study elucidates a novel facet of JB12 and JB14 in that their expression could be regulated in response to stress caused by the presence of ER stressors and the mitochondrial potential uncoupler CCCP. Furthermore, JB14 overexpression could affect the level of PTEN-induced kinase 1 (PINK1) expression under CCCP-mediated stress. Cells with genetic knockout (KO) of DNAJB12 and DNAJB14 exhibited an altered kinetic of phosphorylated Drp1 in response to the stress caused by CCCP treatment. Surprisingly, JB14-KO cells exhibited a prolonged stabilization of PINK1 during chronic exposure to CCCP. Cells depleted with JB12 or JB14 also revealed an increase in the mitochondrial count and branching. Hence, this study indicates the possible novel functions of JB12 and JB14 involving mitochondria in nonstress conditions and under stress caused by CCCP.

J-domain protein chaperone circuits in proteostasis and disease.

The J-domain proteins (JDP) form the largest protein family among cellular chaperones. In cooperation with the Hsp70 chaperone system, these co-chaperones orchestrate a plethora of distinct functions, including those that help maintain cellular proteostasis and development. JDPs evolved largely through the fusion of a J-domain with other protein subdomains. The highly conserved J-domain facilitates the binding and activation of Hsp70s. How JDPs (re)wire Hsp70 chaperone circuits and promote functional diversity remains insufficiently explained. Here, we discuss recent advances in our understanding of the JDP family with a focus on the regulation built around J-domains to ensure correct pairing and assembly of JDP-Hsp70 machineries that operate on different clientele under various cellular growth conditions.

Specification of Hsp70 Function by Hsp40 Co-chaperones.

Cellular homeostasis and stress survival requires maintenance of the proteome and suppression of proteotoxicity. Molecular chaperones promote cell survival through repair of misfolded proteins and cooperation with protein degradation machines to discard terminally damaged proteins. Hsp70 family members play an essential role in cellular protein metabolism by binding and releasing non-native proteins to facilitate protein folding, refolding, and degradation. Hsp40 (DnaJ-like proteins) family members are Hsp70 co-chaperones that determine the fate of Hsp70 clients by facilitating protein folding, assembly, and degradation. Hsp40s select substrates for Hsp70 via use of an intrinsic chaperone activity to bind non-native regions of proteins. During delivery of bound cargo Hsp40s employ a conserved J-domain to stimulate Hsp70 ATPase activity and thereby stabilize complexes between Hsp70 and non-native proteins. This review describes the mechanisms by which different Hsp40s use specialized sub-domains to direct clients of Hsp70 for triage between folding versus degradation.

Lysosome docking to WIPI1 rings and ER-connected phagophores occurs during DNAJB12- and GABARAP-dependent selective autophagy of misfolded P23H-rhodopsin.

We report on how the endoplasmic reticulum (ER)-associated-autophagy pathway (ERAA) delivers P23H-rhodopsin (P23H-R) to the lysosome. P23H-R accumulates in an ERAD-resistant conformation that is stabilized in a detergent-soluble state by DNAJB12 and Hsp70. P23H-R, DNAJB12, and FIP200 colocalize in discrete foci that punctuate the rim of omegasome rings coated by WIPI1. Loss of DNAJB12 function prevents the association of P23H-R containing ER tubules with omegasomes. P23H-R tubules thread through the wall of WIPI1 rings into their central cavity. Transfer of P23H-R from ER-connected phagophores to lysosomes requires GABARAP and is associated with the transient docking of lysosomes to WIPI1 rings. After departure from WIPI1 rings, new patches of P23H-R are seen in the membranes of lysosomes. The absence of GABARAP prevents transfer of P23H-R from phagophores to lysosomes without interfering with docking. These data identify lysosome docking to omegasomes as an important step in the DNAJB12- and GABARAP-dependent autophagic disposal of dominantly toxic P23H-R.

DNAJB12 and Hsp70 Mediate Triage of Misfolded Membrane Proteins for Proteasomal versus Lysosomal Degradation.

The endoplasmic reticulum (ER) fills the cell with a continuous network of sealed membrane tubules and sheets. The ER is subdivided into microdomains mediating one-third of total protein biosynthesis, oxidative protein folding, secretion, protein quality control, calcium signaling, marcoautophagy/autophagy, stress sensing, and apoptosis. Defects in ER-calcium homeostasis underlie several diseases. Damage to the ER by misfolded membrane proteins is suppressed by specific HSPA/Hsp70 and DNAJ/Hsp40 chaperone pairs that select intermediates for ubiquitination and ER-associated degradation (ERAD) via the proteasome. The ER-transmembrane Hsp40 chaperone DNAJB12 and HSPA/Hsp70 also target toxic intermediates of misfolded membrane proteins for ER-associated autophagy (ERAA). DNAJB12-HSPA/Hsp70 maintain membrane protein degradation intermediates in detergent-soluble and degradation-competent states. DNAJB12-HSPA/Hsp70 also interact with the autophagy initiation kinase ULK1 on ER tubules containing ERAD-resistant misfolded membrane proteins (ERAD-RMPs). Omegasomes are ER microdomains where the autophagosome precursor or phagophore (PG) forms. ER tubules loaded with ERAD-RMPs enter omegasomes where they are converted into ER-connected PG (ER-PG). The Atg8 (autophagy related 8)-family member GABARAP (GABA type A receptor-associated protein) facilitates transfer of ERAD-RMPs from ER-PGs to autolysosomes (AL) that dock transiently with omegasomes. This article describes a model for DNAJB12-HSPA/Hsp70 action during the conformation-dependent triage in the ER of misfolded membrane proteins for folding versus proteasomal or AL degradation.

DNAJB12 and Hsp70 triage arrested intermediates of N1303K-CFTR for endoplasmic reticulum-associated autophagy.

The transmembrane Hsp40 DNAJB12 and cytosolic Hsp70 cooperate on the endoplasmic reticulum's (ER) cytoplasmic face to facilitate the triage of nascent polytopic membrane proteins for folding versus degradation. N1303K is a common mutation that causes misfolding of the ion channel CFTR, but unlike F508del-CFTR, biogenic and functional defects in N1303K-CFTR are resistant to correction by folding modulators. N1303K is reported to arrest CFTR folding at a late stage after partial assembly of its N-terminal domains. N1303K-CFTR intermediates are clients of JB12-Hsp70 complexes, maintained in a detergent-soluble state, and have a relatively long 3-h half-life. ER-associated degradation (ERAD)-resistant pools of N1303K-CFTR are concentrated in ER tubules that associate with autophagy initiation sites containing WIPI1, FlP200, and LC3. Destabilization of N1303K-CFTR or depletion of JB12 prevents entry of N1303K-CFTR into the membranes of ER-connected phagophores and traffic to autolysosomes. In contrast, the stabilization of intermediates with the modulator VX-809 promotes the association of N1303K-CFTR with autophagy initiation machinery. N1303K-CFTR is excluded from the ER-exit sites, and its passage from the ER to autolysosomes does not require ER-phagy receptors. DNAJB12 operates in biosynthetically active ER microdomains to triage membrane protein intermediates in a conformation-specific manner for secretion versus degradation via ERAD or selective-ER-associated autophagy.

Biological Characterization of F508delCFTR Protein Processing by the CFTR Corrector ABBV-2222/GLPG2222.

Cystic fibrosis (CF) is the most common monogenic autosomal recessive disease in Caucasians caused by pathogenic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (CFTR). Significant small molecule therapeutic advances over the past two decades have been made to target the defective CFTR protein and enhance its function. To address the most prevalent defect of the defective CFTR protein (i.e., F508del mutation) in CF, two biomolecular activities are required, namely, correctors to increase the amount of properly folded F508delCFTR levels at the cell surface and potentiators to allow the effective opening, i.e., function of the F508delCFTR channel. Combined, these activities enhance chloride ion transport yielding improved hydration of the lung surface and subsequent restoration of mucociliary clearance. To enhance clinical benefits to CF patients, a complementary triple combination therapy consisting of two corrector molecules, type 1 (C1) and type 2, with additive mechanisms along with a potentiator are being investigated in the clinic for maximum restoration of mutated CFTR function. We report the identification and in vitro biologic characterization of ABBV-2222/GLPG2222 (4-[(2R,4R)-4-({[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl}amino)-7-(difluoromethoxy)-3,4-dihydro-2H-chromen-2-yl]benzoic acid),-a novel, potent, and orally bioavailable C1 corrector developed by AbbVie-Galapagos and currently in clinical trials-which exhibits substantial improvements over the existing C1 correctors. This includes improvements in potency and drug-drug interaction (DDI) compared with 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid (VX-809, Lumacaftor) and improvements in potency and efficacy compared with 1-(2,2-difluoro-1,3-benzodioxol-5-yl)-N-[1-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)indol-5-yl]cyclopropane-1-carboxamide (VX-661, Tezacaftor). ABBV-2222/GLPG2222 exhibits potent in vitro functional activity in primary patient cells harboring F508del/F508del CFTR with an EC50 value <10 nM. SIGNIFICANCE STATEMENT: To address the most prevalent defect of the defective CFTR protein (i.e., F508del mutation) in cystic fibrosis, AbbVie-Galapagos has developed ABBV-2222/GLPG2222, a novel, potent, and orally bioavailable C1 corrector of this protein. ABBV-2222/GLPG2222, which is currently in clinical trials, exhibits potent in vitro functional activity in primary patient cells harboring F508del/F508del CFTR and substantial improvements over the existing C1 correctors.

Function, evolution, and structure of J-domain proteins.

Hsp70 chaperone systems are very versatile machines present in nearly all living organisms and in nearly all intracellular compartments. They function in many fundamental processes through their facilitation of protein (re)folding, trafficking, remodeling, disaggregation, and degradation. Hsp70 machines are regulated by co-chaperones. J-domain containing proteins (JDPs) are the largest family of Hsp70 co-chaperones and play a determining role functionally specifying and directing Hsp70 functions. Many features of JDPs are not understood; however, a number of JDP experts gathered at a recent CSSI-sponsored workshop in Gdansk (Poland) to discuss various aspects of J-domain protein function, evolution, and structure. In this report, we present the main findings and the consensus reached to help direct future developments in the field of Hsp70 research.

Endoplasmic reticulum stress-induced degradation of DNAJB12 stimulates BOK accumulation and primes cancer cells for apoptosis.

DNAJB12 (JB12) is an endoplasmic reticulum (ER)-associated Hsp40 family protein that recruits Hsp70 to the ER surface to coordinate the function of ER-associated and cytosolic chaperone systems in protein quality control. Hsp70 is stress-inducible, but paradoxically, we report here that JB12 was degraded by the proteasome during severe ER stress. Destabilized JB12 was degraded by ER-associated degradation complexes that contained HERP, Sel1L, and gp78. JB12 was the only ER-associated chaperone that was destabilized by reductive stress. JB12 knockdown by siRNA led to the induction of caspase processing but not the unfolded protein response. ER stress-induced apoptosis is regulated by the highly labile and ER-associated BCL-2 family member BOK, which is controlled at the level of protein stability by ER-associated degradation components. We found that JB12 was required in human hepatoma cell line 7 (Huh-7) liver cancer cells to maintain BOK at low levels, and BOK was detected in complexes with JB12 and gp78. Depletion of JB12 during reductive stress or by shRNA from Huh-7 cells was associated with accumulation of BOK and activation of Caspase 3, 7, and 9. The absence of JB12 sensitized Huh-7 to death caused by proteotoxic agents and the proapoptotic chemotherapeutic LCL-161. In summary, JB12 is a stress-sensitive Hsp40 whose degradation during severe ER stress provides a mechanism to promote BOK accumulation and induction of apoptosis.

Focal Adhesion Kinase-mediated Phosphorylation of Beclin1 Protein Suppresses Cardiomyocyte Autophagy and Initiates Hypertrophic Growth.

Autophagy is an evolutionarily conserved intracellular degradation/recycling system that is essential for cellular homeostasis but is dysregulated in a number of diseases, including myocardial hypertrophy. Although it is clear that limiting or accelerating autophagic flux can result in pathological cardiac remodeling, the physiological signaling pathways that fine-tune cardiac autophagy are poorly understood. Herein, we demonstrated that stimulation of cardiomyocytes with phenylephrine (PE), a well known hypertrophic agonist, suppresses autophagy and that activation of focal adhesion kinase (FAK) is necessary for PE-stimulated autophagy suppression and subsequent initiation of hypertrophic growth. Mechanistically, we showed that FAK phosphorylates Beclin1, a core autophagy protein, on Tyr-233 and that this post-translational modification limits Beclin1 association with Atg14L and reduces Beclin1-dependent autophagosome formation. Remarkably, although ectopic expression of wild-type Beclin1 promoted cardiomyocyte atrophy, expression of a Y233E phosphomimetic variant of Beclin1 failed to affect cardiomyocyte size. Moreover, genetic depletion of Beclin1 attenuated PE-mediated/FAK-dependent initiation of myocyte hypertrophy in vivo Collectively, these findings identify FAK as a novel negative regulator of Beclin1-mediated autophagy and indicate that this pathway can facilitate the promotion of compensatory hypertrophic growth. This novel mechanism to limit Beclin1 activity has important implications for treating a variety of pathologies associated with altered autophagic flux.

Restoration of R117H CFTR folding and function in human airway cells through combination treatment with VX-809 and VX-770.

Cystic fibrosis (CF) is a lethal recessive genetic disease caused primarily by the F508del mutation in the CF transmembrane conductance regulator (CFTR). The potentiator VX-770 was the first CFTR modulator approved by the FDA for treatment of CF patients with the gating mutation G551D. Orkambi is a drug containing VX-770 and corrector VX809 and is approved for treatment of CF patients homozygous for F508del, which has folding and gating defects. At least 30% of CF patients are heterozygous for the F508del mutation with the other allele encoding for one of many different rare CFTR mutations. Treatment of heterozygous F508del patients with VX-809 and VX-770 has had limited success, so it is important to identify heterozygous patients that respond to CFTR modulator therapy. R117H is a more prevalent rare mutation found in over 2,000 CF patients. In this study we investigated the effectiveness of VX-809/VX-770 therapy on restoring CFTR function in human bronchial epithelial (HBE) cells from R117H/F508del CF patients. We found that VX-809 stimulated more CFTR activity in R117H/F508del HBEs than in F508del/F508del HBEs. R117H expressed exclusively in immortalized HBEs exhibited a folding defect, was retained in the ER, and degraded prematurely. VX-809 corrected the R117H folding defect and restored channel function. Because R117 is involved in ion conductance, VX-770 acted additively with VX-809 to restore CFTR function in chronically treated R117H/F508del cells. Although treatment of R117H patients with VX-770 has been approved, our studies indicate that Orkambi may be more beneficial for rescue of CFTR function in these patients.

From CFTR biology toward combinatorial pharmacotherapy: expanded classification of cystic fibrosis mutations.

More than 2000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) have been described that confer a range of molecular cell biological and functional phenotypes. Most of these mutations lead to compromised anion conductance at the apical plasma membrane of secretory epithelia and cause cystic fibrosis (CF) with variable disease severity. Based on the molecular phenotypic complexity of CFTR mutants and their susceptibility to pharmacotherapy, it has been recognized that mutations may impose combinatorial defects in CFTR channel biology. This notion led to the conclusion that the combination of pharmacotherapies addressing single defects (e.g., transcription, translation, folding, and/or gating) may show improved clinical benefit over available low-efficacy monotherapies. Indeed, recent phase 3 clinical trials combining ivacaftor (a gating potentiator) and lumacaftor (a folding corrector) have proven efficacious in CF patients harboring the most common mutation (deletion of residue F508, ΔF508, or Phe508del). This drug combination was recently approved by the U.S. Food and Drug Administration for patients homozygous for ΔF508. Emerging studies of the structural, cell biological, and functional defects caused by rare mutations provide a new framework that reveals a mixture of deficiencies in different CFTR alleles. Establishment of a set of combinatorial categories of the previously defined basic defects in CF alleles will aid the design of even more efficacious therapeutic interventions for CF patients.

Transcription errors induce proteotoxic stress and shorten cellular lifespan.

Transcription errors occur in all living cells; however, it is unknown how these errors affect cellular health. To answer this question, we monitor yeast cells that are genetically engineered to display error-prone transcription. We discover that these cells suffer from a profound loss in proteostasis, which sensitizes them to the expression of genes that are associated with protein-folding diseases in humans; thus, transcription errors represent a new molecular mechanism by which cells can acquire disease phenotypes. We further find that the error rate of transcription increases as cells age, suggesting that transcription errors affect proteostasis particularly in aging cells. Accordingly, transcription errors accelerate the aggregation of a peptide that is implicated in Alzheimer's disease, and shorten the lifespan of cells. These experiments reveal a previously unappreciated role for transcriptional fidelity in cellular health and aging.

Specification of Hsp70 function by Type I and Type II Hsp40.

Cellular homeostasis and stress survival requires maintenance of the proteome and suppression of proteotoxicity. Molecular chaperones promote cell survival through repair of misfolded proteins and cooperation with protein degradation machines to discard terminally damaged proteins. Hsp70 family members play an essential role in cellular protein metabolism by binding and releasing nonnative proteins to facilitate protein folding, refolding and degradation. Hsp40 family members are Hsp70 co-chaperones that determine the fate of Hsp70 clients by facilitating protein folding, assembly, and degradation. Hsp40s select substrates for Hsp70 via use of an intrinsic chaperone activity to bind non-native regions of proteins. During delivery of bound cargo Hsp40s employ a conserved J-domain to stimulate Hsp70 ATPase activity and thereby stabilize complexes between Hsp70 and non-native proteins. Type I and Type II Hsp40s direct Hsp70 to preform multiple functions in protein homeostasis. This review describes the mechanisms by which Type I and Type II sub-types of Hsp40 bind and deliver substrates to Hsp70.

Kinetics of C-C and C-H bond cleavage in phenyl alkane radical cations generated by photoinduced electron transfer.

Employing nanosecond laser flash photolysis, we determined the relative importance of two fragmentation modes, namely, C-C bond cleavage and deprotonation, for the radical cation of 1,1,2,2-tetraphenylethane photogenerated by electron transfer to cyanoaromatic singlet excited states in acetonitrile at room temperature. Analysis of the kinetic data for this phenyl alkane suggests that the C-C bond cleavage dominates over the deprotonation by a ratio of about 2:1. In addition, the deprotonation kinetics of diphenylmethane, 1,1-diphenylethane, triphenylmethane, and several phenyl-substituted alcohols have been investigated. To aid identification and characterization, experiments based on two laser pulses in tandem (308 and 337.1 nm) were performed to probe fluorescence and photochemistry of the transient radicals formed as products of radical ion fragmentation. The first-order rate constants for growth of transient absorptions due to fragmentation-derived radicals were measured to be ≥1 × 10(6) s(-1). Activation parameters, with activation enthalpies in the range 10-18 kJ/mol and activation entropies between -60 and -91 J/(mol.K), are also reported for fragmentation kinetics of radical cations of several systems under study.

Polyglutamine-rich suppressors of huntingtin toxicity act upstream of Hsp70 and Sti1 in spatial quality control of amyloid-like proteins.

Protein conformational maladies such as Huntington Disease are characterized by accumulation of intracellular and extracellular protein inclusions containing amyloid-like proteins. There is an inverse correlation between proteotoxicity and aggregation, so facilitated protein aggregation appears cytoprotective. To define mechanisms for protective protein aggregation, a screen for suppressors of nuclear huntingtin (Htt103Q) toxicity was conducted. Nuclear Htt103Q is highly toxic and less aggregation prone than its cytosolic form, so we identified suppressors of cytotoxicity caused by Htt103Q tagged with a nuclear localization signal (NLS). High copy suppressors of Htt103Q-NLS toxicity include the polyQ-domain containing proteins Nab3, Pop2, and Cbk1, and each suppresses Htt toxicity via a different mechanism. Htt103Q-NLS appears to inactivate the essential functions of Nab3 in RNA processing in the nucleus. Function of Pop2 and Cbk1 is not impaired by nuclear Htt103Q, as their respective polyQ-rich domains are sufficient to suppress Htt103Q toxicity. Pop2 is a subunit of an RNA processing complex and is localized throughout the cytoplasm. Expression of just the Pop2 polyQ domain and an adjacent proline-rich stretch is sufficient to suppress Htt103Q toxicity. The proline-rich domain in Pop2 resembles an aggresome targeting signal, so Pop2 may act in trans to positively impact spatial quality control of Htt103Q. Cbk1 accumulates in discrete perinuclear foci and overexpression of the Cbk1 polyQ domain concentrates diffuse Htt103Q into these foci, which correlates with suppression of Htt toxicity. Protective action of Pop2 and Cbk1 in spatial quality control is dependent upon the Hsp70 co-chaperone Sti1, which packages amyloid-like proteins into benign foci. Protein:protein interactions between Htt103Q and its intracellular neighbors lead to toxic and protective outcomes. A subset of polyQ-rich proteins buffer amyloid toxicity by funneling toxic aggregation intermediates to the Hsp70/Sti1 system for spatial organization into benign species.

Quality control autophagy degrades soluble ERAD-resistant conformers of the misfolded membrane protein GnRHR.

Molecular chaperones triage misfolded proteins via action as substrate selectors for quality control (QC) machines that fold or degrade clients. Herein, the endoplasmic reticulum (ER)-associated Hsp40 JB12 is reported to participate in partitioning mutant conformers of gonadotropin-releasing hormone receptor (GnRHR), a G protein-coupled receptor, between ER-associated degradation (ERAD) and an ERQC autophagy pathway. ERQC autophagy degrades E90K-GnRHR because pools of its partially folded and detergent-soluble degradation intermediates are resistant to ERAD. S168R-GnRHR is globally misfolded and disposed of via ERAD, but inhibition of p97, the protein retrotranslocation motor, shunts S168R-GnRHR from ERAD to ERQC autophagy. Partially folded and grossly misfolded forms of GnRHR associate with JB12 and Hsp70. Elevation of JB12 promotes ERAD of S168R-GnRHR, with E90K-GnRHR being resistant. E90K-GnRHR elicits association of the Vps34 autophagy initiation complex with JB12. Interaction between ER-associated Hsp40s and the Vps34 complex permits the selective degradation of ERAD-resistant membrane proteins via ERQC autophagy.

Inhibition of post-translational N-glycosylation by HRD1 that controls the fate of ABCG5/8 transporter.

N-glycosylation of proteins in endoplasmic reticulum is critical for protein quality control. We showed here a post-translational N-glycosylation affected by the HRD1 E3 ubiquitin ligase. Both WT- and E3-defective C329S-HRD1 decreased the level of high mannose form of ABCG8, a protein that heterodimerizes with ABCG5 to control sterol balance. Meanwhile, HRD1 increased the non-glycosylated ABCG8 regardless of its E3 activity, thereby suppressing full maturation of ABCG5/8 transporter. Pulse chase and mutational analysis indicated that HRD1 inhibits STT3B-dependent post-translational N-glycosylation of ABCG8. Whereas, HRD1 had only slight effect on the N-glycosylation status of ABCG5; rather it accelerated ABCG5 degradation in an E3 activity-dependent manner. Finally, RMA1, another E3 ubiquitin ligase, accelerated the degradation of both ABCG5 and ABCG8 via E3 activity-dependent manner. HRD1 and RMA1 may therefore be negative regulators of disease-associated transporter ABCG5/ABCG8. The findings also highlight the unexpected E3 activity-independent role of HRD1 in the regulation of N-glycosylation.