A Novel Quality Control Compartment Derived from the Endoplasmic Reticulum

Degradation of proteins that, because of improper or suboptimal processing, are retained in the endoplasmic reticulum (ER) involves retrotranslocation to reach the cytosolic ubiquitin-proteasome machinery. We found that substrates of this pathway, the precursor of human asialoglycoprotein receptor H2a and free heavy chains of murine class I major histocompatibility complex (MHC), accumulate in a novel preGolgi compartment that is adjacent to but not overlapping with the centrosome, the Golgi complex, and the ER-to-Golgi intermediate compartment (ERGIC). On its way to degradation, H2a associated increasingly after synthesis with the ER translocon Sec61. Nevertheless, it remained in the secretory pathway upon proteasomal inhibition, suggesting that its retrotranslocation must be tightly coupled to the degradation process. In the presence of proteasomal inhibitors, the ER chaperones calreticulin and calnexin, but not BiP, PDI, or glycoprotein glucosyltransferase, concentrate in the subcellular region of the novel compartment. The "quality control" compartment is possibly a subcompartment of the ER. It depends on microtubules but is insensitive to brefeldin A. We discuss the possibility that it is also the site for concentration and retrotranslocation of proteins that, like the mutant cystic fibrosis transmembrane conductance regulator, are transported to the cytosol, where they form large aggregates, the "aggresomes."     

ER protein quality control and proteasome-mediated protein degradation

A variety of mutant polypeptides that are associated with human disease are targeted for degradation by an endoplasmic reticulum (ER) quality control system. In addition, physiological signals and viral gene products can target the degradation of several ER resident proteins and secreted proteins passing through the ER. Although the mechanism of protein quality control and the site of degradation were obscure, recent data indicate that degradation requires the cytosolic proteasome. Biochemical and genetic analyses have indicated that both lumenal and integral membrane proteins are selected for proteolysis and exported to the cytosol by a process that in several cases requires ER associated molecular chaperones.     

Glycopeptide export from the endoplasmic reticulum into cytosol is mediated by a mechanism distinct from that for export of misfolded glycoprotein

When glycoproteins formed in the endoplasmic reticulum (ER) are misfolded, they are generally translocated into the cytosol for ubiquitination and are subsequently degraded by the proteasome. This system, the so-called ER-associated glycoprotein degradation, is important for eukaryotes to maintain the quality of glycoproteins generated in the ER. It has been established in yeast that several distinct proteins are involved in this translocation and degradation processes. Small glycopeptides formed in the ER are exported to the cytosol in a similar manner. This glycopeptide export system is conserved from yeast to mammalian cells, suggesting its basic biological significance for eukaryotic cells. These two export systems (for misfolded glycoproteins and glycopeptides) share some properties, such as a requirement for ATP and involvement of Sec61p, a central membrane protein presumably forming a dislocon channel for export of proteins. However, the machinery of glycopeptide export is poorly understood. In this study, various mutants known to have an effect on export/degradation of misfolded glycoproteins were examined for glycopeptide export activity with a newly established assay method. Surprisingly, most of the mutants were found not to exhibit a defect in glycopeptide export. The only gene that was found to be required on efficient export of both types of substrates was PMR1, the gene encoding the medial-Golgi Ca(2+)/Mn(2+)-ion pump. These results provide evidence that although the systems involved in export of misfolded glycoproteins and glycopeptides share some properties, they have exhibited distinct differences.     

Glutathione limits Ero1-dependent oxidation in the endoplasmic reticulum

Many proteins of the secretory pathway contain disulfide bonds that are essential for structure and function. In the endoplasmic reticulum (ER), Ero1 alpha and Ero1 beta oxidize protein disulfide isomerase (PDI), which in turn transfers oxidative equivalents to newly synthesized cargo proteins. However, oxidation must be limited, as some reduced PDI is necessary for disulfide isomerization and ER-associated degradation. Here we show that in semipermeable cells, PDI is more oxidized, disulfide bonds are formed faster, and high molecular mass covalent protein aggregates accumulate in the absence of cytosol. Addition of reduced glutathione (GSH) reduces PDI and restores normal disulfide formation rates. A higher GSH concentration is needed to balance oxidative folding in semipermeable cells overexpressing Ero1 alpha, indicating that cytosolic GSH and lumenal Ero1 alpha play antagonistic roles in controlling the ER redox. Moreover, the overexpression of Ero1 alpha significantly increases the GSH content in HeLa cells. Our data demonstrate tight connections between ER and cytosol to guarantee redox exchange across compartments: a reducing cytosol is important to ensure disulfide isomerization in secretory proteins.     

Activation of the Ras-cAMP signal transduction pathway inhibits the proteasome-independent degradation of misfolded protein aggregates in the endoplasmic reticulum lumen

Many kinds of misfolded secretory proteins are known to be degraded in the endoplasmic reticulum (ER). Dislocation of misfolded proteins from the ER to the cytosol and subsequent degradation by the proteasome have been demonstrated. Using the yeast Saccharomyces cerevisiae, we have been studying the secretion of a heterologous protein, Rhizopus niveus aspartic proteinase-I (RNAP-I). Previously, we found that the pro sequence of RNAP-I is important for the folding and secretion, and that Deltapro, a mutated derivative of RNAP-I in which the entire region of the pro sequence is deleted, forms gross aggregates in the yeast ER. In this study, we show that the degradation of Deltapro occurs independently of the proteasome. Its degradation was not inhibited either by a potent proteasome inhibitor or in a proteasome mutant. We also show that neither the export from the ER nor the vacuolar proteinase is required for the degradation of Deltapro. These results raise the possibility that the Deltapro aggregates are degraded in the ER lumen. We have isolated a yeast mutant in which the degradation of Deltapro is delayed. We show that the mutated gene is IRA2, which encodes a GTPase-activating protein for Ras. Because Ira2 protein is a negative regulator of the Ras-cAMP pathway, this result suggests that hyperactivation of the Ras-cAMP pathway inhibits the degradation of Deltapro. Consistently, down-regulation of the Ras-cAMP pathway in the ira2 mutant suppressed the defect of the degradation of Deltapro. Thus, the Ras-cAMP signal transduction pathway seems to control the proteasome-independent degradation of the ER misfolded protein aggregates.     

Cytosolic aggregates in presence of non‐translocated proteins perturb endoplasmic reticulum structure and dynamics

Presence of cytosolic protein aggregates and membrane damage are two common attributes of neurodegenerative diseases. These aggregates delay degradation of non‐translocated protein precursors leading to their persistence and accumulation in the cytosol. Here, we find that cells with intracellular protein aggregates (of cytosolic prion protein or huntingtin) destabilize the endoplasmic reticulum (ER) morphology and dynamics when non‐translocated protein load is high. This affects trafficking of proteins out from the ER, relative distribution of the rough and smooth ER and three‐way junctions that are essential for the structural integrity of the membrane network. The changes in ER membranes may be due to high aggregation tendency of the ER structural proteins—reticulons, and altered distribution of those associated with the three‐way ER junctions—Lunapark. Reticulon4 is seen to be enriched in the aggregate fractions in presence of non‐translocated protein precursors. This could be mitigated by improving signal sequence efficiencies of the proteins targeted to the ER. These were observed using PrP variants and the seven‐pass transmembrane protein (CRFR1) with different signal sequences that led to diverse translocation efficiencies. This identifies a previously unappreciated consequence of cytosolic aggregates on non‐translocated precursor proteins—their persistent presence affects ER morphology and dynamics. This may be one of the ways in which cytosolic aggregates can affect endomembranes during neurodegenerative disease.     

A microsomal GTPase is required for glycopeptide export from the mammalian endoplasmic reticulum

Bidirectional transport of proteins via the Sec61p translocon across the endoplasmic reticulum (ER) membrane is a recognized component of the ER quality control machinery. Following translocation and engagement by the luminal quality control system, misfolded and unassembled proteins are exported from the ER lumen back to the cytosol for degradation by the proteasome. Additionally, other ER contents, including oligosaccharides, oligopeptides, and glycopeptides, are efficiently exported from mammalian and yeast systems, indicating that bidirectional transport across ER membranes is a general eukaryotic phenomenon. Glycopeptide and protein export from the ER in in vitro systems is both ATP- and cytosol-dependent. Using a well established system to study glycopeptide export and conventional liquid chromatography, we isolated a single polypeptide species of 23 kDa from rat liver cytosol that was capable of fully supporting glycopeptide export from rat microsomes in the presence of an ATP-regenerating system. The protein was identified by mass spectrometric sequence analysis as guanylate kinase (GK), a housekeeping enzyme critical in the regulation of cellular GTP levels. We confirmed the ability of GK to substitute for complete cytosol by reconstitution of glycopeptide export from rat liver microsomes using highly purified recombinant GK from Saccharomyces cerevisiae. Most significantly, we found that the GK (and hence the cytosolic component) requirement was fully bypassed by low micromolar concentrations of GDP or GTP. Similarly, export was inhibited by non-hydrolyzable analogues of GDP and GTP, indicating a requirement for GTP hydrolysis. Membrane integrity was fully maintained under assay conditions, as no ER luminal proteins were released. Competence for glycopeptide export was abolished by very mild protease treatment of microsomes, indicating the presence of an essential protein on the cytosolic face of the ER membrane. These data demonstrate that export of glycopeptide export is controlled by a microsomal GTPase and is independent of cytosolic protein factors.     

Aggresomes: A Cellular Response to Misfolded Proteins

Intracellular deposition of misfolded protein aggregates into ubiquitin-rich cytoplasmic inclusions is linked to the pathogenesis of many diseases. Why these aggregates form despite the existence of cellular machinery to recognize and degrade misfolded protein and how they are delivered to cytoplasmic inclusions are not known. We have investigated the intracellular fate of cystic fibrosis transmembrane conductance regulator (CFTR), an inefficiently folded integral membrane protein which is degraded by the cytoplasmic ubiquitin-proteasome pathway. Overexpression or inhibition of proteasome activity in transfected human embryonic kidney or Chinese hamster ovary cells led to the accumulation of stable, high molecular weight, detergent-insoluble, multiubiquitinated forms of CFTR. Using immunofluorescence and transmission electron microscopy with immunogold labeling, we demonstrate that undegraded CFTR molecules accumulate at a distinct pericentriolar structure which we have termed the aggresome. Aggresome formation is accompanied by redistribution of the intermediate filament protein vimentin to form a cage surrounding a pericentriolar core of aggregated, ubiquitinated protein. Disruption of microtubules blocks the formation of aggresomes. Similarly, inhibition of proteasome function also prevented the degradation of unassembled presenilin-1 molecules leading to their aggregation and deposition in aggresomes. These data lead us to propose that aggresome formation is a general response of cells which occurs when the capacity of the proteasome is exceeded by the production of aggregation-prone misfolded proteins.     

Type 2 transglutaminase is involved in the autophagy-dependent clearance of ubiquitinated proteins

Eukaryotic cells are equipped with an efficient quality control system to selectively eliminate misfolded and damaged proteins, and organelles. Abnormal polypeptides that escape from proteasome-dependent degradation and aggregate in the cytosol can be transported via microtubules to inclusion bodies called 'aggresomes', where misfolded proteins are confined and degraded by autophagy. Here, we show that Type 2 transglutaminase (TG2) knockout mice display impaired autophagy and accumulate ubiquitinated protein aggregates upon starvation. Furthermore, p62-dependent peroxisome degradation is also impaired in the absence of TG2. We also demonstrate that, under cellular stressful conditions, TG2 physically interacts with p62 and they are localized in cytosolic protein aggregates, which are then recruited into autophagosomes, where TG2 is degraded. Interestingly, the enzyme's crosslinking activity is activated during autophagy and its inhibition leads to the accumulation of ubiquitinated proteins. Taken together, these data indicate that the TG2 transamidating activity has an important role in the assembly of protein aggregates, as well as in the clearance of damaged organelles by macroautophagy.     

Efficient and qualitatively distinct MHC class I-restricted presentation of antigen targeted to the endoplasmic reticulum

For most nascent glycoprotein Ags, the MHC class I-restricted processing pathway begins in the endoplasmic reticulum (ER). From this location, they are translocated to the cytosol for degradation by the proteasome. A reasonable assumption is that processing of exocytic Ags is less efficient than that of cytosolic Ags, due to the requirement for additional handling, but that the processing pathways for the two types of proteins are otherwise similar. To test this, we compared the presentation of three epitopes within influenza nucleoprotein (NP) when this Ag is targeted to the cytosol or the ER. Surprisingly, under conditions of limited Ag expression, presentation of two proteasome-dependent epitopes is comparable when NP is targeted to the ER while presentation of a third is negatively impacted. Furthermore, presentation of the third epitope is unaffected by the addition of proteasome inhibitor when cytosolic NP is expressed but is significantly enhanced when exocytic NP is expressed. These results indicate that delivery of Ag to the ER need not preclude efficient presentation and that processing of cytosolic and ER-targeted Ag is qualitatively distinct.     

Divergent regulation of protein synthesis in the cytosol and endoplasmic reticulum compartments of mammalian cells

In eukaryotic cells, mRNAs encoding signal sequence-bearing proteins undergo translation-dependent trafficking to the endoplasmic reticulum (ER), thereby restricting secretory and integral membrane protein synthesis to the ER compartment. However, recent studies demonstrating that mRNAs encoding cytosolic/nucleoplasmic proteins are represented on ER-bound polyribosomes suggest a global role for the ER in cellular protein synthesis. Here, we examined the steady-state protein synthesis rates and compartmental distribution of newly synthesized proteins in the cytosol and ER compartments. We report that ER protein synthesis rates exceed cytosolic protein synthesis rates by 2.5- to 4-fold; yet, completed proteins accumulate to similar levels in the two compartments. These data suggest that a significant fraction of cytosolic proteins undergo synthesis on ER-bound ribosomes. The compartmental differences in steady-state protein synthesis rates correlated with a divergent regulation of the tRNA aminoacylation/deacylation cycle. In the cytosol, two pathways were observed to compete for aminoacyl-tRNAs-protein synthesis and aminoacyl-tRNA hydrolysis-whereas on the ER tRNA deacylation is tightly coupled to protein synthesis. These findings identify a role for the ER in global protein synthesis, and they suggest models where compartmentalization of the tRNA acylation/deacylation cycle contributes to the regulation of global protein synthesis rates.     

Evidence for high-capacity bidirectional glucose transport across the endoplasmic reticulum membrane by genetically encoded fluorescence resonance energy transfer nanosensors

Glucose release from hepatocytes is important for maintenance of blood glucose levels. Glucose-6-phosphate phosphatase, catalyzing the final metabolic step of gluconeogenesis, faces the endoplasmic reticulum (ER) lumen. Thus, glucose produced in the ER has to be either exported from the ER into the cytosol before release into circulation or exported directly by a vesicular pathway. To measure ER transport of glucose, fluorescence resonance energy transfer-based nanosensors were targeted to the cytosol or the ER lumen of HepG2 cells. During perfusion with 5 mM glucose, cytosolic levels were maintained at approximately 80% of the external supply, indicating that plasma membrane transport exceeded the rate of glucose phosphorylation. Glucose levels and kinetics inside the ER were indistinguishable from cytosolic levels, suggesting rapid bidirectional glucose transport across the ER membrane. A dynamic model incorporating rapid bidirectional ER transport yields a very good fit with the observed kinetics. Plasma membrane and ER membrane glucose transport differed regarding sensitivity to cytochalasin B and showed different relative kinetics for galactose uptake and release, suggesting catalysis by distinct activities at the two membranes. The presence of a high-capacity glucose transport system on the ER membrane is consistent with the hypothesis that glucose export from hepatocytes occurs via the cytosol by a yet-to-be-identified set of proteins.     

Distinct steps in dislocation of luminal endoplasmic reticulum-associated degradation substrates: roles of endoplamic reticulum-bound p97/Cdc48p and proteasome

Dislocation of endoplasmic reticulum-associated degradation (ERAD) substrates from the endoplasmic reticulum (ER) lumen to cytosol is considered to occur in a single step that is tightly coupled to proteasomal degradation. Here we show that dislocation of luminal ERAD substrates occurs in two distinct consecutive steps. The first is passage across ER membrane to the ER cytosolic face, where substrates can accumulate as ubiquitin conjugates. In vivo, this step occurs despite proteasome inhibition but requires p97/Cdc48p because substrates remain entrapped in ER lumen and are prevented from ubiquitination in cdc48 yeast strain. The second dislocation step is the release of accumulated substrates to the cytosol. In vitro, this release requires active proteasome, consumes ATP, and relies on salt-removable ER-bound components, among them the ER-bound p97 and ER-bound proteasome, which specifically interact with the cytosol-facing substrates. An additional role for Cdc48p subsequent to ubiquitination is revealed in the cdc48 strain at permissive temperature, consistent with our finding that p97 recognizes luminal ERAD substrates through multiubiquitin. BiP interacts exclusively with ERAD substrates, suggesting a role for this chaperone in ERAD. We propose a model that assigns the cytosolic face of the ER as a midpoint to which luminal ERAD substrates emerge and p97/Cdc48p and the proteasome are recruited. Although p97/Cdc48p plays a dual role in dislocation and is involved both in passage of the substrate across ER membrane and subsequent to its ubiquitination, the proteasome takes part in the release of the substrate from the ER face to the cytosol en route to degradation.     

Intracellular localization of proteasomes

Proteasomes are present in the cytoplasm and in the nuclei of all eukaryotic cells, however their relative abundance within those compartments is highly variable. In the cytoplasm, proteasomes associate with the centrosomes, cytoskeletal networks and the outer surface of the endoplasmic reticulum (ER). In the nucleus, proteasomes are present throughout the nucleoplasm but are void from the nucleoli. Sometimes they associate with discrete subnuclear domains called the PML nuclear bodies (POD domains). PML bodies in the nucleus, and the pericentrosomal area of the cytoplasm may function as proteolytic centers of the cell, since they are enriched in components of the proteasome system. Under conditions of impaired proteolysis proteasomes and ubiquitinated proteins further accumulate at these locations, forming organized aggregates. In case of the pericentrosomal area those aggregates have been termed "aggresomes". Once formed, aggresomes can impair the function of the proteasome system, which may promote apoptosis. Under favorable conditions they can be cleared, probably by autophagy.     

Hsp90-mediated cytosolic refolding of exogenous proteins internalized by dendritic cells

Dendritic cells efficiently internalize exogenous protein antigens by fluid-phase uptake and receptor-mediated endocytosis. Such antigens contribute to cross-presentation by being translocated into the cytosol for proteasomal degradation, which liberates immunogenic peptides that can bind to major histocompatibility complex (MHC) class I molecules after being transported into the endoplasmic reticulum (ER). MHC class I-peptide complexes are then expressed on the cell surface and presented to CD8+ T cells. Here we show that internalized proteins can have an alternative fate. After internalization, proteins are first unfolded to allow translocation into the cytosol using a pathway related to ER-associated degradation (ERAD). Subsequently the unfolded proteins can undergo cytosolic refolding assisted by the chaperone Hsp90. These observations not only clarify the cellular processes regulating cytosolic access following endocytosis, but also demonstrate that functional proteins can potentially regain their activity in the cytosol of dendritic cells.     

Reflux of Endoplasmic Reticulum proteins to the cytosol inactivates tumor suppressors

In the past decades, many studies reported the presence of endoplasmic reticulum (ER)‐resident proteins in the cytosol. However, the mechanisms by which these proteins relocate and whether they exert cytosolic functions remain unknown. We find that a subset of ER luminal proteins accumulates in the cytosol of glioblastoma cells isolated from mouse and human tumors. In cultured cells, ER protein reflux to the cytosol occurs upon ER proteostasis perturbation. Using the ER luminal protein anterior gradient 2 (AGR2) as a proof of concept, we tested whether the refluxed proteins gain new functions in the cytosol. We find that refluxed, cytosolic AGR2 binds and inhibits the tumor suppressor p53. These data suggest that ER reflux constitutes an ER surveillance mechanism to relieve the ER from its contents upon stress, providing a selective advantage to tumor cells through gain‐of‐cytosolic functions—a phenomenon we name ER to Cytosol Signaling (ERCYS).     

Presenilin-binding protein forms aggresomes in monkey kidney COS-7 cells

A novel presenilin-binding protein (PBP) is specifically expressed in the brain, and its level in the soluble fraction of Alzheimer's disease (AD) brains is much less than that in the age-matched controls. Recently, several proteins, including presenilin (PS), have been found to form structures of aggregated proteins, called aggresomes, when the production of the proteins exceeds their rate of degradation by proteasomes. Based on these observations it has been proposed that the aggresome may represent one of the mechanisms forthe formation of cytoplasmic deposits which are linked to the pathogenesis of neurodegenerative disorders including AD. It is shown here that the overexpression of PBP or the suppression of proteasome activity in monkey kidney COS-7 cells leads to the accumulation of detergent-insoluble and multiubiquitinated PBP aggregates. PBP also forms aggregates in primary cultures of neurons in the presence of a proteasome inhibitors. PBP aggregates have the characteristics of aggresomes, including the localization to microtubule organization centers and the disruption of intermediate filaments. These observations suggest that the malfunctioning of the proteasome can cause the formation of PBP aggresomes, which may lead to AD.     

Long Term Aggresome Accumulation Leads to DNA Damage,p53-dependent Cell Cycle Arrest,and Steric Interference in Mitosis

Juxtanuclear aggresomes form in cells when levels of aggregation-prone proteins exceed the capacity of the proteasome to degrade them. It is widely believed that aggresomes have a protective function, sequestering potentially damaging aggregates until these can be removed by autophagy. However, most in-cell studies have been carried out over a few days at most, and there is little information on the long term effects of aggresomes. To examine these long term effects, we created inducible, single-copy cell lines that expressed aggregation-prone polyglutamine proteins over several months. We present evidence that, as perinuclear aggresomes accumulate, they are associated with abnormal nuclear morphology and DNA double-strand breaks, resulting in cell cycle arrest via the phosphorylated p53 (Ser-15)-dependent pathway. Further analysis reveals that aggresomes can have a detrimental effect on mitosis by steric interference with chromosome alignment, centrosome positioning, and spindle formation. The incidence of apoptosis also increased in aggresome-containing cells. These severe defects developed gradually after juxtanuclear aggresome formation and were not associated with small cytoplasmic aggregates alone. Thus, our findings demonstrate that, in dividing cells, aggresomes are detrimental over the long term, rather than protective. This suggests a novel mechanism for polyglutamine-associated developmental and cell biological abnormalities, particularly those with early onset and non-neuronal pathologies.     

Deubiquitinases USP20/33 promote the biogenesis of tail-anchored membrane proteins

Numerous proteins that have hydrophobic transmembrane domains (TMDs) traverse the cytosol and posttranslationally insert into cellular membranes. It is unclear how these hydrophobic membrane proteins evade recognition by the cytosolic protein quality control (PQC), which typically recognizes exposed hydrophobicity in misfolded proteins and marks them for proteasomal degradation by adding ubiquitin chains. Here, we find that tail-anchored (TA) proteins, a vital class of membrane proteins, are recognized by cytosolic PQC and are ubiquitinated as soon as they are synthesized in cells. Surprisingly, the ubiquitinated TA proteins are not routed for proteasomal degradation but instead are handed over to the targeting factor, TRC40, and delivered to the ER for insertion. The ER-associated deubiquitinases, USP20 and USP33, remove ubiquitin chains from TA proteins after their insertion into the ER. Thus, our data suggest that deubiquitinases rescue posttranslationally targeted membrane proteins that are inappropriately ubiquitinated by PQC in the cytosol.     

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

Many aberrant or unassembled proteins synthesized in the endoplasmic reticulum (ER) are degraded by cytosolic proteasomes. To investigate how soluble glycoproteins destined for degradation are retrotranslocated across the ER membrane, we analyzed the fate of two IgM subunits, mu and J, retained in the ER by myeloma cells that do not synthesize light chains. Degradation of mu and J is prevented by proteasome inhibitors, suggesting that both chains are retrotranslocated to be disposed of by proteasomes. Indeed, when proteasomes are inhibited, some deglycosylated J chains that no longer contain intrachain disulfide bonds accumulate in the cytosol. However, abundant glycosylated J chains are still present in the ER at time points in which degradation would have been almost complete in the absence of proteasome inhibitors, suggesting that retrotranslocation and degradation are coupled events. This was confirmed by protease protection and cell fractionation assays, which revealed that virtually all mu chains are retained in the ER lumen in a glycosylated state when proteasomes are inhibited. Association with calnexin correlated with the failure of mu chains to dislocate to the cytosol. Taken together, these results suggest that active proteasomes are required for the extraction of Ig subunits from the ER, though the requirements for retrotranslocation may differ among individual substrates.