Finding the will and the way of ERAD substrate retrotranslocation

doi:10.1016/j.ceb.2012.05.010

Current Opinion in Cell Biology

Volume 24, Issue 4, August 2012, Pages 460-466

https://doi.org/10.1016/j.ceb.2012.05.010 Get rights and content

ER-associated degradation (ERAD) is a mechanism by which numerous ER-localized proteins are targeted for cytosolic degradation by the ubiquitin–proteasome system. A surprising and still-cryptic requirement of this process is the energy dependent retrotranslocation of both lumenal and membrane-embedded ER proteins into the cytosol for ongoing ubiquitination and proteasomal destruction. The current understanding, results, and open questions are discussed below for this intriguing and critical process of ERAD.

Section snippets

ERAD and retrotranslocation

ER (endoplasmic reticulum)-associated protein degradation (ERAD) is a general term for the proteolytic pathways that degrade numerous ER proteins, including both luminal and integral membrane substrates [1, 2]. (We hesitate to use the term ‘clients’ for the substrates, since except for some members of Wall Street, most operations do not function to destroy their clients…). ERAD is primarily involved in protein quality control, since most ERAD substrates are damaged or misfolded proteins. ERAD

Retrotranslocation of all classes of ERAD substrates

Retrotranslocation was discovered in the Wolf group's pioneering studies of DER genes responsible for ER degradation of the prototype ERAD substrate CPY*. The normally vacuole-bound CPY* is retained and degraded in the ER by virtue of a point mutation that renders it unfoldable. The reasonable model that the entirely lumenal CPY* was degraded within the ER was shattered by the stunning discovery that DER2 encoded the cytoplasmic ubiquitin E2 known as Ubc7 [7^••], indicating that lumenal CPY* was

Retrotranslocation employs the Cdc48/p97 AAA-ATPase

The ‘will’ or the energy requirements for retrotranslocation are unknown. However, current thinking is that removal of ER proteins might be expected to require cellular energy. For luminal substrates, if ER exit is analogous to ER entry, then complete unfolding of the substrate would be required, although this requirement is not clear. Experiments using strongly folded luminal domains such as DHFR appended to ERAD substrates to test the requirement for unfolding have provided diverse results [24

Way-ing in on egress: how do ERAD substrates leave the ER?

The old saying goes, “where there's a will, there's a way” and ERAD is no exception: if Cdc48/p97 provides the energetic ‘will’, then the way would be the actual route across and out of the ER membrane. This aspect of the ERAD remains unclear despite much activity from many groups. Because the usual way to get polypeptide into the ER is to employ a protein channel, it is reasonable to imagine that this is also the way proteins exit the ER. There have been a variety of candidates suggested and

Sec61 anterograde channel as an exit route

In the quest to find the ‘ERAD channel’, an early and reasonable suggestion was that the same channel used for ER entry, Sec61 and its partner subunits, was also employed for the similar task of moving polypeptides the other direction. Consistent with this idea, small peptides have been observed to exit the ER by this route [43], and mammalian Sec61α has been reported to associate with some ERAD substrates [44], proteasomes [45], and the HRD ERAD ligase complex [46, 47]. Unfortunately, because

Derlin: an alDERnative channel for retrotranslocation

A second channel candidate arose from proteomic studies of the viral US11 complex that orchestrates the extremely efficient degradation of single-spanning MHC-I molecules. They revealed a family of ER-localized candidate channel proteins known as derlins [53••, 54••], homologous to the very first ERAD gene ever identified, DER1, which is the yeast version of this family [55]. This connection between the mammalian and yeast studies implies a broadly conserved role for these factors in ERAD.

ERAD E3 ligases as channel components

A third class of pore candidates is suggested from ERAD E3 ligase topology. The two principal ERAD E3 ligases, Hrd1 and Doa10 both have large multispanning membrane domains (Hrd1: 6 spans, Doa10: 14 spans [56, 57]). Furthermore, the Hrd1 transmembrane domain has a high proportion of hydrophilic residues, as might be expected to line a peptide-transporting pore [58^••]. These features have led to the appealing idea that in addition to ubiquitination, these E3s might provide a retrotranslocation

Towards an integrated picture of retrotranslocation

The collection of often ambiguous results described above may be providing clues about features of retrotranslocation, and resolution of those issues will be important steps in understanding the mechanism of this transport pathway. It may be that the lone still-undiscovered channel is a ‘small’ genetic target, and thus still awaiting discovery by assiduious genetic means. Another possibility for the lack of consensus on a channel is that we are dealing with the ‘Escape from New York’ scenario:

Lipid dynamics in retrotranslocation

Alternatively, it may be that there are no retrotranslocation channels, and that a totally novel route is employed to remove ERAD substrates to the cytosol. It has been suggested that the machinery that forms lipid droplets, which are derived from the ER outer leaflet and end up on the cytosol, might provide a non-channel mechanism for dislocation, at least for membrane proteins [67]. However, it is clear that at least in yeast the loss of lipid droplet formation by removal of the appropriate

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

• of special interest
•• of outstanding interest

Acknowledgements

We (RYH and TS) wish to thank members of our research groups past and present for discussions, data, and down-home fun. In addition, we dedicate this article to the memory of Steve Jobs, to whom nearly every biological scientist owes a debt of gratitude for powerful, friendly and whimsical computing tools of all stripe. RYH is supported by NIH grant DK051996, and by an ARRA supplement to that award. TS is generously supported by the Deutsche Forschungsgemeinschaft.

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Disulfide-crosslink analysis of the ubiquitin ligase Hrd1 complex during endoplasmic reticulum-associated protein degradation
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Misfolded proteins in the lumen of the endoplasmic reticulum (ER) are retrotranslocated into the cytosol and degraded by the ubiquitin-proteasome system, a pathway termed luminal ER-associated protein degradation. Retrotranslocation is mediated by a conserved protein complex, consisting of the ubiquitin ligase Hrd1 and four associated proteins (Der1, Usa1, Hrd3, and Yos9). Photocrosslinking experiments provided preliminary evidence for the polypeptide path through the membrane but did not reveal specific interactions between amino acids in the substrate and Hrd1 complex. Here, we have used site-specific disulfide crosslinking to map the interactions of a glycosylated model substrate with the Hrd1 complex in live S. cerevisiae cells. Together with available electron cryo-microscopy structures, the results show that the substrate interacts on the luminal side with both a groove in Hrd3 and the lectin domain of Yos9 and inserts a loop into the membrane, with one side of the loop interacting with the lateral gate of Der1 and the other with the lateral gate of Hrd1. Our disulfide crosslinking experiments also show that two Hrd1 molecules can interact through their lateral gates and that Hrd1 autoubiquitination is required for the disassembly of these Hrd1 dimers. Taken together, these data define the path of a polypeptide through the ER membrane and suggest that autoubiquitination of inactive Hrd1 dimers is required to generate active Hrd1 monomers.
Endoplasmic Reticulum Stress in Disease
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Proper protein folding in the endoplasmic reticulum (ER) is subject to alterations in the extracellular, as well as the intracellular environment. The accumulation of misfolded proteins in the ER activates the unfolded protein response (UPR), a collection of adaptive signaling pathways that are essential to maintain protein-folding homeostasis in all eukaryotic cells. The UPR reduces protein influx into the ER, increases ER folding capacity, and directs misfolded proteins to degradation. If protein misfolding is not resolved, cells activate death pathways. Protein misfolding in the ER contributes to the etiologies of many diseases and targeting the UPR may provide novel therapies for many pathologies.
Asaronic acid inhibits ER stress sensors and boosts functionality of ubiquitin-proteasomal degradation in 7β-hydroxycholesterol-loaded macrophages
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Misfolded proteins are formed in the endoplasmic reticulum (ER) due to diverse stimuli including oxidant production, calcium disturbance, and inflammatory factors. Accumulation of these non-native proteins in the ER evokes cellular stress involving the activation of unfolded protein response (UPR) and the execution of ER-associated degradation (ERAD). Naturally-occurring plant compounds are known to interfere with UPR due to their antioxidant and anti-inflammatory activities, leading to inhibition of ER stress. However, there are few studies dealing with the protective effects of natural compounds on the functionality of ERAD.
The current study examined whether asaronic acid enhanced ubiquitin-proteasomal degradation in J774A.1 murine macrophages exposed to 7β-hydroxycholesterol, a risk factor for atherosclerosis. Asaronic acid (2,4,5-trimethoxybenzoic acid), identified as one of purple perilla constituents, has anti-diabetic and anti-inflammatory effects. Little is known regarding the effects of asaronic acid on the ERAD process and the ubiquitin-proteasomal degradation.
Murine macrophages were incubated with 28 μM 7β-hydroxycholesterol in absence and presence of 1–20 μΜ asaronic acid for up to 24 h. Nontoxic asaronic acid in macrophage diminished the activation of the ER stress sensors of ATF6, IRE1 and PERK stimulated by 7β-hydroxycholesterol. This methoxybenzoic acid down-regulated the oxysterol-induced expression of EDEM1, OS9, Sel1L-Hrd1 and p97/VCP1, all required for the recognition, recruitment and dislocation of misfolded proteins. On the other hand, asaronic acid enhanced the ubiquitin-proteasomal degradation of non-native proteins dislocated to the cytosol by 7β-hydroxycholesterol, which entailed the induction of the chaperones of Hsp70 and CHIP and the increased colocalization of ubiquitin and proteasomes. Taken together, asaronic acid attenuated the induction of the UPR-associated sensors and the dislocation-linked transmembrane components in the ER. Conversely, this compound enhanced the proteasomal degradation of dislocated non-native proteins in concert with the chaperones of Hsp70 and CHIP through ubiquitination.
These observations demonstrate that asaronic acid may be a potent atheroprotective agent as a natural chaperone targeting ER stress-associated macrophage injury.
Arabidopsis HIPP proteins regulate endoplasmic reticulum-associated degradation of CKX proteins and cytokinin responses
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Citation Excerpt :
Given that the CKX1 domain mediating the interaction with HIPPs is localized in the luminal part of the protein (Figure 1 and Supplemental Figure 4C), BiFC between CKX1 and HIPP7 implicates that the CKX1 protein in the detected complex represents a form which was relocated to the cytosolic side of the membrane. This is consistent with the recent finding that CKX1 is targeted to the ERAD pathway (Niemann et al., 2015), which requires retrotranslocation of target proteins from the ER prior to delivery to the cytosolic/nuclear proteasome (Hampton and Sommer, 2012). To test whether CKX1–HIPP7 interaction involves the retrotranslocated CKX1 protein, we performed BiFC assays using CKX1421DLVK and CKX1409 that were unable to interact with HIPPs in yeast.
Eukaryotic organisms are equipped with quality-control mechanisms that survey protein folding in the endoplasmic reticulum (ER) and remove non-native proteins by ER-associated degradation (ERAD). Recent research has shown that cytokinin-degrading CKX proteins are subjected to ERAD during plant development. The mechanisms of plant ERAD, including the export of substrate proteins from the ER, are not fully understood, and the molecular components involved in the ERAD of CKX are unknown. Here, we show that heavy metal-associated isoprenylated plant proteins (HIPPs) interact specifically with CKX proteins synthesized in the ER and processed by ERAD. CKX–HIPP protein complexes were detected at the ER as well as in the cytosol, suggesting that the complexes involve retrotranslocated CKX protein species. Altered CKX levels in HIPP-overexpressing and higher-order hipp mutant plants suggest that the studied HIPPs control the ERAD of CKX. Deregulation of CKX proteins caused corresponding changes in the cytokinin signaling activity and triggered typical morphological cytokinin responses. Notably, transcriptional repression of HIPP genes by cytokinin indicates a feedback regulatory mechanism of cytokinin homeostasis and signaling responses. Moreover, loss of function of HIPP genes constitutively activates the unfolded protein response and compromises the ER stress tolerance. Collectively, these results suggests that HIPPs represent novel functional components of plant ERAD.
Derlin rhomboid pseudoproteases employ substrate engagement and lipid distortion to enable the retrotranslocation of ERAD membrane substrates
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Nearly one-third of proteins are initially targeted to the endoplasmic reticulum (ER) membrane, where they are correctly folded and then delivered to their final cellular destinations. To prevent the accumulation of misfolded membrane proteins, ER-associated degradation (ERAD) moves these clients from the ER membrane to the cytosol, a process known as retrotranslocation. Our recent work in Saccharomyces cerevisiae reveals a derlin rhomboid pseudoprotease, Dfm1, is involved in the retrotranslocation of ubiquitinated ERAD membrane substrates. In this study, we identify conserved residues of Dfm1 that are critical for retrotranslocation. We find several retrotranslocation-deficient Loop 1 mutants that display impaired binding to membrane substrates. Furthermore, Dfm1 possesses lipid thinning function to facilitate in the removal of ER membrane substrates, and this feature is conserved in its human homolog, Derlin-1, further implicating that derlin-mediated retrotranslocation is a well-conserved process.

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Finding the will and the way of ERAD substrate retrotranslocation

Section snippets

ERAD and retrotranslocation

Retrotranslocation of all classes of ERAD substrates

Retrotranslocation employs the Cdc48/p97 AAA-ATPase

Way-ing in on egress: how do ERAD substrates leave the ER?

Sec61 anterograde channel as an exit route

Derlin: an alDERnative channel for retrotranslocation

ERAD E3 ligases as channel components

Towards an integrated picture of retrotranslocation

Lipid dynamics in retrotranslocation

References and recommended reading

Acknowledgements

J Biol Chem

Genetics

Proc Natl Acad Sci USA

Crit Rev Biochem Mol Biol

Science

Nature

Mol Biol Cell

EMBO J

Nat Cell Biol

Mol Cell Biol

Nat Struct Biol

J Biol Chem

J Biol Chem

Nature

J Cell Sci

EMBO J

Nature

J Biol Chem

J Biol Chem

FEBS Lett

Mol Biol Cell

Mol Biol Cell

Nat Cell Biol

Mol Cell

Road to ruin: targeting proteins for degradation in the endoplasmic reticulum

Science

ERAD ubiquitin ligases: multifunctional tools for protein quality control and waste disposal in the endoplasmic reticulum

Bioessays

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

Nat Cell Biol

A conserved ubiquitin ligase of the nuclear envelope/endoplasmic reticulum that functions in both ER-associated and Matalpha2 repressor degradation

Genes Dev

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

Proc Natl Acad Sci USA

The endoplasmic reticulum-associated Hsp40 DNAJB12 and Hsc70 cooperate to facilitate RMA1 E3-dependent degradation of nascent CFTRDeltaF508

Mol Biol Cell

ER degradation of a misfolded luminal protein by the cytosolic ubiquitin–proteasome pathway

Science

Re-entering the translocon from the lumenal side of the endoplasmic reticulum. Studies on mutated carboxypeptidase yscY species

FEBS Lett

The requirement for molecular chaperones during endoplasmic reticulum-associated protein degradation demonstrates that protein export and import are mechanistically distinct

J Biol Chem

Degradation of unassembled soluble Ig subunits by cytosolic proteasomes: evidence that retrotranslocation and degradation are coupled events

FASEB J

Degradation of CFTR by the ubiquitin–proteasome pathway

Cell

Protein quality control as a strategy for cellular regulation: lessons from ubiquitin-mediated regulation of the sterol pathway

Chem Rev