Endoplasmic reticulum-associated degradation (ERAD) is a mechanism during which native and misfolded proteins are recognized and retrotranslocated across the ER membrane to the cytosol for degradation by the ubiquitin-proteasome system. Like other cellular pathways, the factors required for ERAD have been analyzed using both conventional genetic and biochemical approaches. More recently, however, an integrated top-down approach has identified a functional network that underlies the ERAD system. In turn, bottom-up reconstitution has become increasingly sophisticated and elucidated the molecular mechanisms underlying substrate recognition, ubiquitylation, retrotranslocation, and degradation. In addition, a live cell imaging technique and a site-specific in vivo photo-crosslinking approach have further dissected specific steps during ERAD. These technical developments have revealed an unexpected dynamicity of the membrane-associated ERAD complex. In this article, we will discuss how these technical developments have improved our understanding of the ERAD pathway and have led to new questions.
The vacuole in the yeast Saccharomyces cerevisiae plays a number of essential roles, and to provide some of these required functions the vacuole harbors at least seven distinct proteases. These proteases exhibit a range of activities and different classifications, and they follow unique paths to arrive at their ultimate, common destination in the cell. This review will first summarize the major functions of the yeast vacuole and delineate how proteins are targeted to this organelle. We will then describe the specific trafficking itineraries and activities of the characterized vacuolar proteases, and outline select features of a new member of this protease ensemble. Finally, we will entertain the question of why so many proteases evolved and reside in the vacuole, and what future research challenges exist in the field.
protease; S. cerevisiae; hydrolysis; autophagy; endocytosis; metalloprotease; Vps10; CPY; secretory pathway; Pff1
All newly synthesized proteins are subject to quality control check-points, which prevent aberrant polypeptides from harming the cell. For proteins that ultimately reside in the cytoplasm, components that also reside in the cytoplasm were known for many years to mediate quality control. Early biochemical and genetic data indicated that misfolded proteins were selected by molecular chaperones and then targeted to the proteasome (in eukaryotes) or to proteasome-like particles (in bacteria) for degradation. What was less clear was how secreted and integral membrane proteins, which in eukaryotes enter the endoplasmic reticulum (ER), were subject to quality control decisions. In this review, we highlight early studies that ultimately led to the discovery that secreted and integral membrane proteins also utilize several components that constitute the cytoplasmic quality machinery. This component of the cellular quality control pathway is known as ER associated degradation, or ERAD.
The systematic and complete characterization of the Saccharomyces cerevisiae genome and proteome has been stalled in some cases by misannotated genes. One such gene is YBR074W, which was initially annotated as two independent open reading frames (ORFs). We now report on Ybr074, a metalloprotease family member that was initially predicted to reside in the endoplasmic reticulum (ER). Therefore, we tested the hypothesis that Ybr074 may be an ER quality control protease. Instead, indirect immunofluorescence images indicate that Ybr074 is a vacuolar protein, and by employing protease protection assays, we demonstrate that a conserved M28 metalloprotease domain is oriented within the lumen. Involvement of Ybr074 in ER protein quality control was ruled out by examining the stabilities of several well-characterized substrates in strains lacking Ybr074. Finally, using a proteomic approach, we show that disrupting Ybr074 function affects the levels of select factors implicated in vacuolar trafficking and osmoregulation. Together, our data indicate that Ybr074 is the only multi-spanning vacuolar membrane protease found in the yeast Saccharomyces cerevisiae.
The Kir2.1 potassium channel is targeted by endoplasmic reticulum–associated degradation in yeast. To identify other Kir2.1 quality control factors, a novel yeast screen was performed. ESCRT components were among the strongest hits from the screen. Consistent with these data, ESCRT also regulates Kir2.1 stability in human cells.
Protein quality control (PQC) is required to ensure cellular health. PQC is recognized for targeting the destruction of defective polypeptides, whereas regulated protein degradation mechanisms modulate the concentration of specific proteins in concert with physiological demands. For example, ion channel levels are physiologically regulated within tight limits, but a system-wide approach to define which degradative systems are involved is lacking. We focus on the Kir2.1 potassium channel because altered Kir2.1 levels lead to human disease and Kir2.1 restores growth on low-potassium medium in yeast mutated for endogenous potassium channels. Using this system, first we find that Kir2.1 is targeted for endoplasmic reticulum–associated degradation (ERAD). Next a synthetic gene array identifies nonessential genes that negatively regulate Kir2.1. The most prominent gene family that emerges from this effort encodes members of endosomal sorting complex required for transport (ESCRT). ERAD and ESCRT also mediate Kir2.1 degradation in human cells, with ESCRT playing a more prominent role. Thus multiple proteolytic pathways control Kir2.1 levels at the plasma membrane.
New polyomaviruses are continually being identified, and it is likely that links between this virus family and disease will continue to emerge. Unfortunately, a specific treatment for polyomavirus-associated disease is lacking. Because polyomaviruses express large Tumor Antigen, TAg, we hypothesized that small molecule inhibitors of the essential ATPase activity of TAg would inhibit viral replication. Using a new screening platform, we identified inhibitors of TAg's ATPase activity. Lead compounds were moved into a secondary assay, and ultimately two FDA approved compounds, bithionol and hexachlorophene, were identified as the most potent TAg inhibitors known to date. Both compounds inhibited Simian Virus 40 replication as assessed by plaque assay and quantitative PCR. Moreover, these compounds inhibited BK virus, which causes BKV Associated Nephropathy. In neither case was host cell viability compromised at these concentrations. Our data indicate that directed screening for TAg inhibitors is a viable method to identify polyomavirus inhibitors, and that bithionol and hexachlorophene represent lead compounds that may be further modified and/or ultimately used to combat diseases associated with polyomavirus infection.
polyomavirus; bithionol; hexachlorophene; T antigen; molecular chaperone; high throughput screen
Protein disulfide isomerases (PDIs) are conserved chaperone-like proteins that play an essential role during protein folding and in some cases during degradation. Substrate-specific effects of PDI family members occur during the ER-associated degradation of diverse substrates in yeast and mammalian cells.
ER-associated degradation (ERAD) rids the early secretory pathway of misfolded or misprocessed proteins. Some members of the protein disulfide isomerase (PDI) family appear to facilitate ERAD substrate selection and retrotranslocation, but a thorough characterization of PDIs during the degradation of diverse substrates has not been undertaken, in part because there are 20 PDI family members in mammals. PDIs can also exhibit disulfide redox, isomerization, and/or chaperone activity, but which of these activities is required for the ERAD of different substrate classes is unknown. We therefore examined the fates of unique substrates in yeast, which expresses five PDIs. Through the use of a yeast expression system for apolipoprotein B (ApoB), which is disulfide rich, we discovered that Pdi1 interacts with ApoB and facilitates degradation through its chaperone activity. In contrast, Pdi1's redox activity was required for the ERAD of CPY* (a misfolded version of carboxypeptidase Y that has five disulfide bonds). The ERAD of another substrate, the alpha subunit of the epithelial sodium channel, was Pdi1 independent. Distinct effects of mammalian PDI homologues on ApoB degradation were then observed in hepatic cells. These data indicate that PDIs contribute to the ERAD of proteins through different mechanisms and that PDI diversity is critical to recognize the spectrum of potential ERAD substrates.
The most frequent cause of α1-antitrypsin (here referred to as AT) deficiency is homozygosity for the AT-Z allele, which encodes AT-Z. Such individuals are at increased risk for liver disease due to the accumulation of aggregation-prone AT-Z in the endoplasmic reticulum of hepatocytes. However, the penetrance and severity of liver dysfunction in AT deficiency is variable, indicating that unknown genetic and environmental factors contribute to its occurrence. There is evidence that the rate of AT-Z degradation may be one such contributing factor. Through the use of several AT-Z model systems, it is now becoming appreciated that AT-Z can be degraded through at least two independent pathways. One model system that has contributed significantly to our understanding of the AT-Z disposal pathway is the yeast, Saccharomyces cerevisiae.
antitrypsin; endoplasmic reticulum–associated degradation; autophagy; yeast
AdaSGC binds Hsc70s to inhibit ATPase activity. Using single-turnover assays, adaSGC, a soluble SGC mimic, preferentially inhibited Hsp40-activated Hsc70 ATP hydrolysis (Ki ~ 10 μM) to reduce C-terminal Hsc70-peptide binding and, potentially, chaperone function. ERAD of misfolded ΔF508 CFTR requires Hsc70-Hsp40 chaperones. In transfected baby hamster kidney (BHK) cells, adaSGC increased ΔF508CFTR ERAD escape, and after low-temperature glycerol rescue, maturation, and iodide efflux. Inhibition of SGC biosynthesis reduced ΔF508CFTR but not wtCFTR expression, whereas depletion of other glycosphingolipids had no affect. WtCFTR transfected BHK cells showed increased SGC synthesis compared with ΔF508CFTR/mock-transfected cells. Partial rescue of ΔF508CFTR by low-temperature glycerol increased SGC synthesis. AdaSGC also increased cellular endogenous SGC levels. SGC in the lung, liver, and kidney was severely depleted in ΔF508CFTR compared with wtCFTR mice, suggesting a role for CFTR in SGC biosynthesis.
Approximately one-third of all newly translated polypeptides interact with the endoplasmic reticulum (ER), an event that is essential to target these nascent proteins to distinct compartments within the cell or to the extracellular milieu. Thus, the ER houses molecular chaperones that augment the folding of this diverse group of macromolecules. The ER also houses the enzymes that catalyze a multitude of posttranslational modifications. If, however, proteins misfold or are improperly modified in the ER they are proteolyzed via a process known as ER-associated degradation (ERAD). During ERAD, substrates are selected by molecular chaperones and chaperone-like proteins. They are then delivered to the cytoplasmic proteasome and hydrolyzed. In most cases, delivery and protea-some-targeting require the covalent attachment of ubiquitin. The discovery and underlying mechanisms of the ERAD pathway have been aided by the development of in vitro assays that employ components derived from the yeast, Saccharomyces cerevisiae. These assays recapitulate the selection of ERAD substrates, the “retrotranslocation” of selected polypeptides from the ER into the cytoplasm, and the proteasome-mediated degradation of the substrate. The ubiquitination of integral membrane ERAD substrates has also been reconstituted.
The 26S proteasome is responsible for most regulated protein turnover and for the degradation of aberrant proteins in eukaryotes. The assembly of this ~2.5 MDa multicatalytic protease requires several dedicated chaperones and, once assembled, substrate selectivity is mediated by ubiquitin conjugation. After modification with ubiquitin, substrates are escorted to the proteasome by myriad factors, including Cdc48 (cell-division cycle 48). Cdc48 also associates with numerous cofactors, but, to date, it is unclear whether each cofactor facilitates proteasome delivery. We discovered that yeast lacking a conserved Cdc48 cofactor, Vms1 [VCP (valosin-containing protein)/Cdc48-associated mitochondrial stress-responsive], accumulate proteasome-targeted ubiquitinated proteins. Vms1 mutant cells also contain elevated levels of unassembled 20S proteasome core particles and select 19S cap subunits. In addition, we found that the ability of Vms1 to support 26S proteasome assembly requires Cdc48 interaction, and that the loss of Vms1 reduced 26S proteasome levels and cell viability after prolonged culture in the stationary phase. The results of the present study highlight an unexpected link between the Cdc48–Vms1 complex and the preservation of proteasome architecture, and indicate how perturbed proteasome assembly affects the turnover of ubiquitinated proteins and maintains viability in aging cells.
cell-division cycle 48 (Cdc48); chaperone; p97; proteasome; ubiquitin; yeast
Heat shock protein 70 (Hsp70) is an important emerging cancer target whose inhibition may affect multiple cancer-associated signaling pathways and, moreover, result in significant cancer cell apoptosis. Despite considerable interest from both academia and pharmaceutical companies in the discovery and development of druglike Hsp70 inhibitors, little success has been reported so far. Here we describe structure–activity relationship studies in the first rationally designed Hsp70 inhibitor class that binds to a novel allosteric pocket located in the N-terminal domain of the protein. These 2,5′-thiodipyrimidine and 5-(phenylthio)-pyrimidine acrylamides take advantage of an active cysteine embedded in the allosteric pocket to act as covalent protein modifiers upon binding. The study identifies derivatives 17a and 20a, which selectively bind to Hsp70 in cancer cells. Addition of high nanomolar to low micromolar concentrations of these inhibitors to cancer cells leads to a reduction in the steady-state levels of Hsp70-sheltered oncoproteins, an effect associated with inhibition of cancer cell growth and apoptosis. In summary, the described scaffolds represent a viable starting point for the development of druglike Hsp70 inhibitors as novel anticancer therapeutics.
Heat shock protein 70 (Hsp70) is
an important emerging cancer target
whose inhibition may affect multiple cancer-associated signaling pathways
and, moreover, result in significant cancer cell apoptosis. Despite
considerable interest from both academia and pharmaceutical companies
in the discovery and development of druglike Hsp70 inhibitors, little
success has been reported so far. Here we describe structure–activity
relationship studies in the first rationally designed Hsp70 inhibitor
class that binds to a novel allosteric pocket located in the N-terminal
domain of the protein. These 2,5′-thiodipyrimidine and 5-(phenylthio)pyrimidine
acrylamides take advantage of an active cysteine embedded in the allosteric
pocket to act as covalent protein modifiers upon binding. The study
identifies derivatives 17a and 20a, which
selectively bind to Hsp70 in cancer cells. Addition of high nanomolar
to low micromolar concentrations of these inhibitors to cancer cells
leads to a reduction in the steady-state levels of Hsp70-sheltered
oncoproteins, an effect associated with inhibition of cancer cell
growth and apoptosis. In summary, the described scaffolds represent
a viable starting point for the development of druglike Hsp70 inhibitors
as novel anticancer therapeutics.
This study describes new yeast expression systems for each subunit of the heterotrimeric epithelial sodium channel (ENaC). We found that a significant amount of each subunit resides in the ER and is destroyed via ERAD. We also found that the chaperone requirements for ENaC subunit degradation were unlike any other ERAD substrate examined.
The epithelial sodium channel (ENaC) is composed of a single copy of an α-, β-, and γ-subunit and plays an essential role in water and salt balance. Because ENaC assembles inefficiently after its insertion into the ER, a substantial percentage of each subunit is targeted for ER-associated degradation (ERAD). To define how the ENaC subunits are selected for degradation, we developed novel yeast expression systems for each ENaC subunit. Data from this analysis suggested that ENaC subunits display folding defects in more than one compartment and that subunit turnover might require a unique group of factors. Consistent with this hypothesis, yeast lacking the lumenal Hsp40s, Jem1 and Scj1, exhibited defects in ENaC degradation, whereas BiP function was dispensable. We also discovered that Jem1 and Scj1 assist in ENaC ubiquitination, and overexpression of ERdj3 and ERdj4, two lumenal mammalian Hsp40s, increased the proteasome-mediated degradation of ENaC in vertebrate cells. Our data indicate that Hsp40s can act independently of Hsp70 to select substrates for ERAD.
A single GAG codon deletion in the gene encoding torsinA is linked to most cases of early-onset torsion dystonia. TorsinA is an ER-localized membrane-associated ATPase from the AAA+ superfamily with an unknown biological function. We investigated the formation of oligomeric complexes of torsinA in cultured mammalian cells and found that wild type torsinA associates into a complex with a molecular weight consistent with that of a homohexamer. Interestingly, the dystonia-linked variant torsinAΔE displayed a reduced propensity to form the oligomers compared to the wild type protein. We also discovered that the deletion of the N-terminal membrane-associating region of torsinA abolished oligomer formation. Our results demonstrate that the dystonia-linked mutation in the torsinA gene produces a protein variant that is deficient in maintaining its oligomeric state and suggest that ER membrane association is required to stabilize the torsinA complex.
Early-onset dystonia, TorsinA; AAA+ ATPase; Protein association
Secretory and membrane proteins that fail to fold in the endoplasmic reticulum (ER) are retained and may be sorted for ER-associated degradation (ERAD). During ERAD, ER-associated components such as molecular chaperones and lectins recognize folding intermediates and specific oligosaccharyl modifications on ERAD substrates. Substrates selected for ERAD are then targeted for ubiquitin- and proteasome-mediated degradation. Because the catalytic steps of the ubiquitin–proteasome system reside in the cytoplasm, soluble ERAD substrates that reside in the ER lumen must be retrotranslocated back to the cytoplasm prior to degradation. In contrast, it has been less clear how polytopic, integral membrane substrates are delivered to enzymes required for ubiquitin conjugation and to the proteasome. In this review, we discuss recent studies addressing how ERAD substrates are recognized, ubiquitinated and delivered to the proteasome and then survey current views of how soluble and integral membrane substrates may be retrotranslocated.
degradation; ER; glycosylation; molecular chaperone; proteasome; proteolysis; transport; ubiquitin
Hsp110s are divergent relatives of Hsp70 chaperones that hydrolyze ATP. Hsp110s serve as Hsp70 nucleotide exchange factors and act directly to maintain polypeptide solubility. To date, the impact of peptide binding on Hsp110 ATPase activity is unknown and an Hsp110/peptide affinity has not been measured. We now report on a peptide that binds to the yeast Hsp110, Sse1p, with a KD of ~2 nM. Surprisingly, the binding of this peptide fails to stimulate Sse1p ATP hydrolysis. Moreover, an Hsp70-binding peptide is unable to associate with Sse1p, suggesting that Hsp70s and Hsp110s possess partially distinct peptide recognition motifs.
Hsp70; molecular chaperone; nucleotide exchange factor; fluorescence; ATPase
The Hsp70 molecular chaperones are ATPases that play critical roles in the pathogenesis of many human diseases, including breast cancer. Hsp70 ATP hydrolysis is relatively weak, but is stimulated by J domain-containing proteins. We identified pyrimidinone-peptoid hybrid molecules that inhibit cell proliferation with greater potency than previously described Hsp70 modulators. In many cases, anti-proliferative activity correlated with inhibition of J domain stimulation of Hsp70.
An increasing body of data links endoplasmic reticulum (ER) function to autophagy. Not surprisingly, then, some aberrant proteins in the ER can be destroyed either via ER associated degradation (ERAD), which is proteasome-mediated, or via autophagy. One such substrate is the “Z” variant of the alpha-1 protease inhibitor (A1Pi), variably known as A1Pi-Z or AT-Z (“anti-trypsin, Z variant”). The wild type protein is primarily synthesized in the liver and is secreted. In contrast, AT-Z, like other ERAD substrates, is retro-translocated from the ER and delivered to the proteasome. However, AT-Z can form high molecular weight polymers that are degraded via autophagy, and cells that accumulate AT-Z polymers ultimately succumb, which leads to liver disease. Therefore, identifying genes that have an impact AT-Z turnover represents an active area of research. To this end, a yeast expression system for AT-Z has proven valuable. For example, a recent study using this system indicates that the activity of a proteasome assembly chaperone (PAC) is critical for maximal AT-Z turnover, which suggests a new role for PACs. Because PACs are conserved, it will be critical to analyze whether these dedicated chaperones are implicated in other diseases associated with ERAD and autophagy.
anti-trypsin; A1Pi; proteasome; chaperone; proteasome assembly chaperone; PAC; yeast; unfolded protein response; liver disease; apoptosis
It remains unclear how misfolded membrane proteins are selected and destroyed during endoplasmic reticulum associated degradation (ERAD). For example, chaperones are thought to solubilize aggregation-prone motifs, and some data suggest that these proteins are degraded at the ER. To better define how membrane proteins are destroyed, the ERAD of Ste6p*, a twelve transmembrane protein, was reconstituted. We found that specific Hsp70/40s act before ubiquitination and facilitate Ste6p* association with an E3 ubiquitin ligase, suggesting an active role for chaperones. Furthermore, polyubiquitination was a prerequisite for retro-translocation, which required the Cdc48 complex and ATP. Surprisingly, the substrate was soluble, and extraction was independent of a ubiquitin chain extension enzyme (Ufd2p). However, Ufd2p increased the degree of ubiquitination and facilitated degradation. These data indicate that polytopic membrane proteins can be extracted from the ER, and define the point of action of chaperones and the requirement for Ufd2p during membrane protein quality control.
molecular chaperone; Hsp70; ubiquitination; Cdc48/p97; Ufd2; proteasome
Most proteins in the secretory pathway are translated, folded, and subjected to quality control at the endoplasmic reticulum (ER). These processes must be flexible enough to process diverse protein conformations, yet specific enough to recognize when a protein should be degraded. Molecular chaperones are responsible for this decision making process. ER associated chaperones assist in polypeptide translocation, protein folding, and ER associated degradation (ERAD). Nevertheless, we are only beginning to understand how chaperones function, how they are recruited to specific substrates and assist in folding/degradation, and how unique chaperone classes make quality control “decisions.”
heat shock proteins; ERAD; proteasome; lectin; degradation
Antitrypsin deficiency is a primary cause of juvenile liver disease, and it arises from expression of the “Z” variant of the α-1 protease inhibitor (A1Pi). Whereas A1Pi is secreted from the liver, A1PiZ is retrotranslocated from the endoplasmic reticulum (ER) and degraded by the proteasome, an event that may offset liver damage. To better define the mechanism of A1PiZ degradation, a yeast expression system was developed previously, and a gene, ADD66, was identified that facilitates A1PiZ turnover. We report here that ADD66 encodes an ∼30-kDa soluble, cytosolic protein and that the chymotrypsin-like activity of the proteasome is reduced in add66Δ mutants. This reduction in activity may arise from the accumulation of 20S proteasome assembly intermediates or from qualitative differences in assembled proteasomes. Add66p also seems to be a proteasome substrate. Consistent with its role in ER-associated degradation (ERAD), synthetic interactions are observed between the genes encoding Add66p and Ire1p, a transducer of the unfolded protein response, and yeast deleted for both ADD66 and/or IRE1 accumulate polyubiquitinated proteins. These data identify Add66p as a proteasome assembly chaperone (PAC), and they provide the first link between PAC activity and ERAD.
Secreted proteins that fail to achieve their native conformations, such as cystic fibrosis transmembrane conductance regulator (CFTR) and particularly the ΔF508-CFTR variant can be selected for endoplasmic reticulum (ER)-associated degradation (ERAD) by molecular chaperones. Because the message corresponding to HSP26, which encodes a small heat-shock protein (sHsp) in yeast was up-regulated in response to CFTR expression, we examined the impact of sHsps on ERAD. First, we observed that CFTR was completely stabilized in cells lacking two partially redundant sHsps, Hsp26p and Hsp42p. Interestingly, the ERAD of a soluble and a related integral membrane protein were unaffected in yeast deleted for the genes encoding these sHsps, and CFTR polyubiquitination was also unaltered, suggesting that Hsp26p/Hsp42p are not essential for polyubiquitination. Next, we discovered that ΔF508-CFTR degradation was enhanced when a mammalian sHsp, αA-crystallin, was overexpressed in human embryonic kidney 293 cells, but wild-type CFTR biogenesis was unchanged. Because αA-crystallin interacted preferentially with ΔF508-CFTR and because purified αA-crystallin suppressed the aggregation of the first nucleotide-binding domain of CFTR, we suggest that sHsps maintain the solubility of ΔF508-CFTR during the ERAD of this polypeptide.
Protein folding is a complex, error-prone process that often results in an irreparable protein by-product. These by-products can be recognized by cellular quality control machineries and targeted for proteasome-dependent degradation. The folding of proteins in the secretory pathway adds another layer to the protein folding “problem,” as the endoplasmic reticulum maintains a unique chemical environment within the cell. In fact, a growing number of diseases are attributed to defects in secretory protein folding, and many of these by-products are targeted for a process known as endoplasmic reticulum-associated degradation (ERAD). Since its discovery, research on the mechanisms underlying the ERAD pathway has provided new insights into how ERAD contributes to human health during both normal and diseases states. Links between ERAD and disease are evidenced from the loss of protein function as a result of degradation, chronic cellular stress when ERAD fails to keep up with misfolded protein production, and the ability of some pathogens to coopt the ERAD pathway. The growing number of ERAD substrates has also illuminated the differences in the machineries used to recognize and degrade a vast array of potential clients for this pathway. Despite all that is known about ERAD, many questions remain, and new paradigms will likely emerge. Clearly, the key to successful disease treatment lies within defining the molecular details of the ERAD pathway and in understanding how this conserved pathway selects and degrades an innumerable cast of substrates.