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.     

In vivo action of the HRD ubiquitin ligase complex: mechanisms of endoplasmic reticulum quality control and sterol regulation

Ubiquitination is used to target both normal proteins for specific regulated degradation and misfolded proteins for purposes of quality control destruction. Ubiquitin ligases, or E3 proteins, promote ubiquitination by effecting the specific transfer of ubiquitin from the correct ubiquitin-conjugating enzyme, or E2 protein, to the target substrate. Substrate specificity is usually determined by specific sequence determinants, or degrons, in the target substrate that are recognized by the ubiquitin ligase. In quality control, however, a potentially vast collection of proteins with characteristic hallmarks of misfolding or misassembly are targeted with high specificity despite the lack of any sequence similarity between substrates. In order to understand the mechanisms of quality control ubiquitination, we have focused our attention on the first characterized quality control ubiquitin ligase, the HRD complex, which is responsible for the endoplasmic reticulum (ER)-associated degradation (ERAD) of numerous ER-resident proteins. Using an in vivo cross-linking assay, we directly examined the association of the separate HRD complex components with various ERAD substrates. We have discovered that the HRD ubiquitin ligase complex associates with both ERAD substrates and stable proteins, but only mediates ubiquitin-conjugating enzyme association with ERAD substrates. Our studies with the sterol pathway-regulated ERAD substrate Hmg2p, an isozyme of the yeast cholesterol biosynthetic enzyme HMG-coenzyme A reductase (HMGR), indicated that the HRD complex discerns between a degradation-competent "misfolded" state and a stable, tightly folded state. Thus, it appears that the physiologically regulated, HRD-dependent degradation of HMGR is effected by a programmed structural transition from a stable protein to a quality control substrate.     

A complex of Yos9p and the HRD ligase integrates endoplasmic reticulum quality control into the degradation machinery

A quality-control system surveys the lumen of the endoplasmic reticulum for terminally misfolded proteins. Polypeptides singled out by this system are ultimately degraded by the cytosolic ubiquitin-proteasome pathway. Key components of both the endoplasmic reticulum quality-control system and the degradation machinery have been identified, but a connection between the two systems has remained elusive. Here, we report an association between the endoplasmic reticulum quality-control lectin Yos9p and Hrd3p, a component of the ubiquitin-proteasome system that links these pathways. We identify designated regions in the luminal domain of Hrd3p that interact with Yos9p and the ubiquitin ligase Hrd1p. Binding of misfolded proteins occurs through Hrd3p, suggesting that Hrd3p recognises proteins that deviate from their native conformation, whereas Yos9p ensures that only terminally misfolded polypeptides are degraded.     

OS-9 and GRP94 deliver mutant alpha1-antitrypsin to the Hrd1-SEL1L ubiquitin ligase complex for ERAD

Terminally misfolded or unassembled proteins in the early secretory pathway are degraded by a ubiquitin- and proteasome-dependent process known as ER-associated degradation (ERAD). How substrates of this pathway are recognized within the ER and delivered to the cytoplasmic ubiquitin-conjugating machinery is unknown. We report here that OS-9 and XTP3-B/Erlectin are ER-resident glycoproteins that bind to ERAD substrates and, through the SEL1L adaptor, to the ER-membrane-embedded ubiquitin ligase Hrd1. Both proteins contain conserved mannose 6-phosphate receptor homology (MRH) domains, which are required for interaction with SEL1L, but not with substrate. OS-9 associates with the ER chaperone GRP94 which, together with Hrd1 and SEL1L, is required for the degradation of an ERAD substrate, mutant alpha(1)-antitrypsin. These data suggest that XTP3-B and OS-9 are components of distinct, partially redundant, quality control surveillance pathways that coordinate protein folding with membrane dislocation and ubiquitin conjugation in mammalian cells.     

Stringent requirement for HRD1, SEL1L,and OS-9/XTP3-B for disposal of ERAD-LS substrates

Sophisticated quality control mechanisms prolong retention of protein-folding intermediates in the endoplasmic reticulum (ER) until maturation while sorting out terminally misfolded polypeptides for ER-associated degradation (ERAD). The presence of structural lesions in the luminal, transmembrane, or cytosolic domains determines the classification of misfolded polypeptides as ERAD-L, -M, or -C substrates and results in selection of distinct degradation pathways. In this study, we show that disposal of soluble (nontransmembrane) polypeptides with luminal lesions (ERAD-L_S substrates) is strictly dependent on the E3 ubiquitin ligase HRD1, the associated cargo receptor SEL1L, and two interchangeable ERAD lectins, OS-9 and XTP3-B. These ERAD factors become dispensable for degradation of the same polypeptides when membrane tethered (ERAD-L_M substrates). Our data reveal that, in contrast to budding yeast, tethering of mammalian ERAD-L substrates to the membrane changes selection of the degradation pathway.     

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.     

A novel mammalian endoplasmic reticulum ubiquitin ligase homologous to the yeast Hrd1

The yeast hHrd1 is a ubiquitin-protein ligase (E3) involved in ER-associated degradation. It was originally identified by genetic methods as an E3 of the yeast cholesterol biosynthetic enzyme HMG-CoA reductase (HMGR). We report the identification and cloning of a human homologue of Hrd1 (hHrd1). Immunofluorescence imaging confirms that the endogenous hHrd1 resides in the ER and in vitro assay demonstrates that it has a ubiquitin-ligase activity. However, the homology between the human and yeast Hrd1 is limited to the N-terminal domain of the proteins, and hHrd1 does not appear to be involved in the degradation of mammalian HMGR.     

SEL1L protein critically determines the stability of the HRD1-SEL1L endoplasmic reticulum-associated degradation (ERAD) complex to optimize the degradation kinetics of ERAD substrates

The mammalian HRD1-SEL1L complex provides a scaffold for endoplasmic reticulum (ER)-associated degradation (ERAD), thereby connecting luminal substrates for ubiquitination at the cytoplasmic surface after their retrotranslocation through the endoplasmic reticulum membrane. In this study the stability of the mammalian HRD1-SEL1L complex was assessed by performing siRNA-mediated knockdown of each of its components. Although endogenous SEL1L is a long-lived protein, the half-life of SEL1L was greatly reduced when HRD1 is silenced. Conversely, transiently expressed SEL1L was rapidly degraded but was stabilized when HRD1 was coexpressed. This was in contrast to the yeast Hrd1p-Hrd3p, where Hrd1p is destabilized by the depletion of Hrd3p, the SEL1L homologue. Endogenous HRD1-SEL1L formed a large ERAD complex (Complex I) associating with numerous ERAD components including ERAD lectin OS-9, membrane-spanning Derlin-1/2, VIMP, and Herp, whereas transiently expressed HRD1-SEL1L formed a smaller complex (Complex II) that was associated with OS-9 but not with Derlin-1/2, VIMP, or Herp. Despite its lack of stable association with the latter components, Complex II supported the retrotranslocation and degradation of model ERAD substrates α1-antitrypsin null Hong-Kong (NHK) and its variant NHK-QQQ lacking the N-glycosylation sites. NHK-QQQ was rapidly degraded when SEL1L was transiently expressed, whereas the simultaneous transfection of HRD1 diminished that effect. SEL1L unassociated with HRD1 was degraded by the ubiquitin-proteasome pathway, which suggests the involvement of a ubiquitin-ligase other than HRD1 in the rapid degradation of both SEL1L and NHK-QQQ. These results indicate that the regulation of the stability and assembly of the HRD1-SEL1L complex is critical to optimize the degradation kinetics of ERAD substrates.     

BiP and calreticulin form an abundant complex that is independent of endoplasmic reticulum stress

BiP is found in association with calreticulin, both in the presence and absence of endoplasmic reticulum stress. Although the BiP-calreticulin complex can be disrupted by ATP, several properties suggest that the calreticulin associated with BiP is neither unfolded nor partially or improperly folded. (1) The complex is stable in vivo and does not dissociate during 8 hr of chase. (2) When present in the complex, calreticulin masks epitopes at the C terminus of BiP that are not masked when BiP is bound to an assembly-defective protein. And (3) overproduction of calreticulin does not lead to the recruitment of more BiP into complexes with calreticulin. The BiP-calreticulin complex can be disrupted by low pH but not by divalent cation chelators. When the endoplasmic reticulum retention signal of BiP is removed, complex formation with calreticulin still occurs, and this explains the poor secretion of the truncated molecule. Gel filtration experiments showed that BiP and calreticulin are present in distinct high molecular weight complexes in which both molecules interact with each other. The possible functions of this complex are discussed.     

Peculiar protein-protein interactions of the novel endoplasmic reticulum membrane protein Rcr1 and ubiquitin ligase Rsp5

Overproduction of the ER membrane protein Rcr1 makes Saccharomyces cerevisiae resistant to Congo red by reducing the chitin content through a unknown mechanism. By both co-immunoprecipitation and yeast two-hybrid experiments, specific interaction between Rcr1 and the ubiquitin ligase Rsp5 was found. This binding was largely mediated by a singular VPEY sequence in Rcr1 in addition to PPSY, the consensus ligand motif of the WW domains. Mutant analysis indicated that Rsp5 and other Rcr1-interacting proteins discovered in the current screen were not engaged in Congo red resistance.     

Interactions between the quality control ubiquitin ligase CHIP and ubiquitin conjugating enzymes

Background  

Ubiquitin (E3) ligases interact with specific ubiquitin conjugating (E2) enzymes to ubiquitinate particular substrate proteins. As the combination of E2 and E3 dictates the type and biological consequence of ubiquitination, it is important to understand the basis of specificity in E2:E3 interactions. The E3 ligase CHIP interacts with Hsp70 and Hsp90 and ubiquitinates client proteins that are chaperoned by these heat shock proteins. CHIP interacts with two types of E2 enzymes, UbcH5 and Ubc13-Uev1a. It is unclear, however, why CHIP binds these E2 enzymes rather than others, and whether CHIP interacts preferentially with UbcH5 or Ubc13-Uev1a, which form different types of polyubiquitin chains.     

Golgi-mediated vacuolar sorting of the endoplasmic reticulum chaperone BiP may play an active role in quality control within the secretory pathway

Quality control in the endoplasmic reticulum (ER) prevents the arrival of incorrectly or incompletely folded proteins at their final destinations and targets permanently misfolded proteins for degradation. Such proteins have a high affinity for the ER chaperone BiP and are finally degraded via retrograde translocation from the ER lumen back to the cytosol. This ER-associated protein degradation (ERAD) is currently thought to constitute the main disposal route, but there is growing evidence for a vacuolar role in quality control. We show that BiP is transported to the vacuole in a wortmannin-sensitive manner in tobacco (Nicotiana tabacum) and that it could play an active role in this second disposal route. ER export of BiP occurs via COPII-dependent transport to the Golgi apparatus, where it competes with other HDEL receptor ligands. When HDEL-mediated retrieval from the Golgi fails, BiP is transported to the lytic vacuole via multivesicular bodies, which represent the plant prevacuolar compartment. We also demonstrate that a subset of BiP-ligand complexes is destined to the vacuole and differs from those likely to be disposed of via the ERAD pathway. Vacuolar disposal could act in addition to ERAD to maximize the efficiency of quality control in the secretory pathway.     

A role for BiP as an adjustor for the endoplasmic reticulum stress-sensing protein Ire1

In the unfolded protein response, the type I transmembrane protein Ire1 transmits an endoplasmic reticulum (ER) stress signal to the cytoplasm. We previously reported that under nonstressed conditions, the ER chaperone BiP binds and represses Ire1. It is still unclear how this event contributes to the overall regulation of Ire1. The present Ire1 mutation study shows that the luminal domain possesses two subregions that seem indispensable for activity. The BiP-binding site was assigned not to these subregions, but to a region neighboring the transmembrane domain. Phenotypic comparison of several Ire1 mutants carrying deletions in the indispensable subregions suggests these subregions are responsible for multiple events that are prerequisites for activation of the overall Ire1 proteins. Unexpectedly, deletion of the BiP-binding site rendered Ire1 unaltered in ER stress inducibility, but hypersensitive to ethanol and high temperature. We conclude that in the ER stress-sensory system BiP is not the principal determinant of Ire1 activity, but an adjustor for sensitivity to various stresses.     

Aberrant quality control in the endoplasmic reticulum impairs the biosynthesis of pulmonary surfactant in mice expressing mutant BiP

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) induces the unfolded protein response (UPR), which alleviates protein overload in the secretory pathway. Although the UPR is activated under diverse pathological conditions, its physiological role during development and in adulthood has not been fully elucidated. Binding immunoglobulin protein (BiP) is an ER chaperone, which is central to ER function. We produced knock-in mice expressing a mutant BiP lacking the retrieval sequence to cause a defect in ER function without completely eliminating BiP. In embryonic fibroblasts, the UPR compensated for mutation of BiP. However, neonates expressing mutant BiP suffered respiratory failure due to impaired secretion of pulmonary surfactant by alveolar type II epithelial cells. Expression of surfactant protein (SP)-C was reduced and the lamellar body was malformed, indicating that BiP plays a critical role in the biosynthesis of pulmonary surfactant. Because pulmonary surfactant requires extensive post-translational processing in the secretory pathway, these findings suggest that in secretory cells, such as alveolar type II cells, the UPR is essential for managing the normal physiological ER protein overload that occurs during development. Moreover, failure of this adaptive mechanism may increase pulmonary susceptibility to environmental insults, such as hypoxia and ischemia, ultimately leading to neonatal respiratory failure.     

Quantity and quality control of gastric proton pump in the endoplasmic reticulum by ubiquitin/proteasome system

The gastric proton pump, H(+),K(+)-ATPase, consists of the catalytic alpha-subunit and the noncatalytic beta-subunit. These subunits are assembled in the endoplasmic reticulum (ER) and leave the ER to reach to the cell surface as a functional holoenzyme. We studied the quantity control mechanism of the H(+),K(+)-ATPase in the ER by using a heterologous expression system in human embryonic kidney 293 cells. The alpha-subunit in the alpha-expressing cells was degraded more rapidly than in the alpha+beta-expressing cells. It was stabilized, however, in the presence of a proteasome inhibitor, lactacystin. Polyubiquitination of the alpha-subunit was observed in the alpha-expressing cells as well as in the alpha+beta-expressing cells. The extent of polyubiquitination was higher in the former alpha-expressing cells especially in the presence of lactacystin. On the other hand, polyubiquitination of the beta-subunit was not observed in the absence and presence of lactacystin. When the alpha-subunit was coexpressed with a mutant beta-subunit that lacks alpha/beta assembly capacity, degradation of the alpha-subunit was accelerated in parallel with increased polyubiquitination of the alpha-subunit. These results indicate that the ubiquitin/proteasome system is involved in degradation of the unassembled alpha-subunits in the ER to control the cell surface expression of the functional alpha/beta holoenzymes.     

RNF170 protein, an endoplasmic reticulum membrane ubiquitin ligase, mediates inositol 1,4,5-trisphosphate receptor ubiquitination and degradation

Inositol 1,4,5-trisphosphate (IP(3)) receptors are endoplasmic reticulum membrane calcium channels that, upon activation, are degraded via the ubiquitin-proteasome pathway. While searching for novel mediators of IP(3) receptor processing, we discovered that RNF170, an uncharacterized RING domain-containing protein, associates rapidly with activated IP(3) receptors. RNF170 is predicted to have three membrane-spanning helices, is localized to the ER membrane, and possesses ubiquitin ligase activity. Depletion of endogenous RNF170 by RNA interference inhibited stimulus-induced IP(3) receptor ubiquitination, and degradation and overexpression of a catalytically inactive RNF170 mutant suppressed stimulus-induced IP(3) receptor processing. A substantial proportion of RNF170 is constitutively associated with the erlin1/2 (SPFH1/2) complex, which has been shown previously to bind to IP(3) receptors immediately after their activation. Depletion of RNF170 did not affect the binding of the erlin1/2 complex to stimulated IP(3) receptors, whereas erlin1/2 complex depletion inhibited RNF170 binding. These results suggest a model in which the erlin1/2 complex recruits RNF170 to activated IP(3) receptors where it mediates IP(3) receptor ubiquitination. Thus, RNF170 plays an essential role in IP(3) receptor processing via the ubiquitin-proteasome pathway.     

Protein folding and quality control in the endoplasmic reticulum

The endoplasmic reticulum (ER) is a highly versatile protein factory that is equipped with chaperones and folding enzymes essential for protein folding. ER quality control guided by these chaperones is essential for life. Whereas correctly folded proteins are exported from the ER, misfolded proteins are retained and selectively degraded. At least two main chaperone classes, BiP and calnexin/calreticulin, are active in ER quality control. Folding factors usually are found in complexes. Recent work emphasises more than ever that chaperones act in concert with co-factors and with each other.     

Marchf6 E3 ubiquitin ligase critically regulates endoplasmic reticulum stress,ferroptosis, and metabolic homeostasis in POMC neurons

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