NEW EMBO MEMBER'S REVIEW. For whom the bell tolls: protein quality control of the endoplasmic reticulum and the ubiquitin±proteasome connection - PDF

The EMBO Journal Vol. 22 No. 10 pp. 2309±2317, 2003 NEW EMBO MEMBER'S REVIEW For whom the bell tolls: protein quality control of the endoplasmic reticulum and the ubiquitin±proteasome connection Zlatka

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The EMBO Journal Vol. 22 No. 10 pp. 2309±2317, 2003 NEW EMBO MEMBER'S REVIEW For whom the bell tolls: protein quality control of the endoplasmic reticulum and the ubiquitin±proteasome connection Zlatka Kostova and Dieter H.Wolf 1 Institut fuèr Biochemie, UniversitaÈt Stuttgart, Pfaffenwaldring 55, Stuttgart, Germany 1 Corresponding author The surveillance of the structural delity of the proteome is of utmost importance to all cells. The endoplasmic reticulum (ER) is the organelle responsible for proper folding and delivery of proteins to the secretory pathway. It contains a sophisticated protein proofreading and elimination mechanism. Failure of this machinery leads to disease and, nally, to cell death. Elimination of misfolded proteins requires retrograde transport across the ER membrane and depends on the central cytoplasmic proteolytic machinery involved in cellular regulation: the ubiquitin±proteasome system. The basics of this process as well as recent advances in the eld are reviewed. Keywords: chaperones/erad/proteasome/ubiquitin Introduction It is of great importance for the cell to regulate the individual entities of its proteome as well as to control the structural delity of each of its members. Proteins destined for secretion, the plasma membrane or the cell surface are translocated from the cytoplasm into the endoplasmic reticulum (ER), the central organelle for further delivery of these proteins to their site of action. Since proteins are translocated into the ER in an unfolded state, it is the primary function of this organelle to modify and fold the translocated proteins to acquire their biologically active conformation (Haigh and Johnson, 2002). In the ER, proteins undergo a quality control procedure that discriminates between properly folded proteins and terminally misfolded species as well as unassembled protein subunits (Ellgaard et al., 1999). The misfolded polypeptides and orphan subunits are subsequently subjected to ER-associated degradation (ERAD). The ERAD process requires retrotranslocation of the malfolded proteins across the ER membrane into the cytoplasm and subsequent degradation by the 26S proteasome (Sommer and Wolf, 1997; Brodsky and McCracken, 1999; Plemper and Wolf, 1999). ER degradation contributes to the molecular pathogenesis of many loss- and gain-of-toxic-function disorders (Aridor and Hannan, 2000; Kostova and Wolf, 2002; Rutishauser and Spiess, 2002). A detailed knowledge of this process is thus of great importance not only for our understanding of this basic cellular mechanism but also for the development of new strategies to treat a diverse set of diseases. Protein folding and the unfolded protein response Many proteins synthesized in the cytoplasm are transported via the secretory pathway to other compartments of the cell, the plasma membrane, or the extracellular space. Secretory proteins are rst translocated into the ER in an unfolded state via an aqueous channel, the Sec61 translocon. In the ER lumen, proteins are properly folded, assembled into multisubunit complexes and covalently modi ed by a large array of ER-resident chaperones and enzymes, before they can enter the secretory pathway (Glick, 2002; Haigh and Johnson, 2002). The major components of this process in the ER are signal peptidase, which cleaves off the signal peptide from the newly translocated proteins; the oligosaccaryl-transferase complex (OST) which carries out N-glycosylation; and protein disul de isomerase (PDI), which participates in disul de bond formation. The two most studied examples of chaperones that assist proteins in their folding are the Hsp70 chaperone BiP (Kar2p in yeast), which recognizes hydrophobic patches on proteins, and calnexin, which binds carbohydrate moieties. Proteins are allowed to exit the ER and enter the secretory pathway only when they are properly folded and modi ed (Ellgaard et al., 1999). The importance of proper folding before ER exit is demonstrated by the existence of an unfolded protein response (UPR), a system that controls the level of the auxiliary folding proteins (Sidrauski et al., 2002). In Saccharomyces cerevisiae, the concentration of unfolded proteins in the ER is sensed by Ire1p, a transmembrane kinase localized to the ER/nuclear envelope, by virtue of a competition between the Ire1p lumenal domain and unfolded proteins for binding to Kar2p (BiP). Depletion of free Kar2p (BiP) molecules due to sequestration by increasing amounts of unfolded proteins leads to the dimerization of Ire1p, a conformational change that transmits a signal across the membrane and activates the cytoplasmic kinase activity. The kinase induces a non-canonical splicing of the HAC1 mrna, allowing synthesis of the Hac1p transcription factor, which upregulates genes containing a UPR response element. This cascade of events leads to an increase in the levels of proteins required for folding and quality control (Travers et al., 2000; Sidrauski et al., 2002). The UPR and ER degradation are tightly interconnected. Overexpression or accumulation of unfolded proteins due to the absence of a component of the ER degradation machinery induces the UPR (Knop et al., 1996a) which, in turn, upregulates the overall level of components of the ER degradation machinery (FriedlaÈnder et al., 2000; Travers et al., 2000). Under normal conditions, cells are able to cope with the amount of naturally present unfolded proteins and do not require induction of the UPR. However, when the level of misfolded proteins ã European Molecular Biology Organization 2309 Z.Kostova and D.H.Wolf Fig. 1. Components of ER quality control and degradation in yeast. rises above a certain level, the UPR becomes essential. In yeast, loss of function of components of both systems is lethal (FriedlaÈnder et al., 2000; Travers et al., 2000). ER quality control Prion diseases like Scrapie (sheep), bovine spongiform encephalopathy (BSE, cattle), or Creutzfeldt±Jakob disease (CJD, human), and other neurodegenerative diseases such as Parkinson and Alzheimer, are the result of precipitated protein aggregates. On the other hand, human diseases such as cystic brosis and lung emphysema are caused by the rapid disappearance of crucial proteins, like the cystic brosis transmembrane conductance regulator (CFTR) and a-1-antitrypsin, respectively (Jensen et al., 1995; Ward et al., 1995; Qu et al., 1996; Kostova and Wolf, 2002; Rutishauser and Spiess, 2002). The discovery of a degradation process for mutated and misfolded ER proteins in yeast (Sommer and Wolf, 1997; Brodsky and McCracken, 1999; Plemper and Wolf, 1999; Kostova and Wolf, 2002) has shed light on the molecular mechanism underlying such seemingly different diseases. Protein function depends greatly on a precise threedimensional conformation, achieved following an uninterrupted, competent folding and assembly process. Mistakes are, however, a fact of life. Mutations leading to an incorrect nal structure result in inactive proteins and, if not properly dealt with, in protein aggregates. To minimize folding mistakes, a complex chaperone system has evolved to assist the folding process and to prevent dead-end conformations (Kopito, 2000). This folding machinery is especially active in the ER, into which nearly all proteins enter in an unfolded state. Protein stretches that leave the Sec61 translocon are immediately occupied by chaperones, preventing hydrophobic surfaces from creating erroneous intra- and/or intermolecular contacts. However, even chaperone-assisted folding cannot guarantee perfection, especially when faced with a mutant protein. To overcome this problem, a quality control system has evolved in the ER to function as a checkpoint that detects improperly folded proteins and targets them for elimination (Ellgaard et al., 1999). The most in uential ER folding components are the Hsp70 chaperone BiP (Kar2p), the lectin like chaperones calnexin and calreticulin, and enzymes involved in disul de bond formation, such as PDI and oxidoreductase Erp57. All of these components also participate in ER quality control. Quite unexpectedly, the elimination of misfolded proteins from the ER is dependent on the cytoplasmic ubiquitin±proteasome system and, therefore, requires protein retrotranslocation (dislocation) across the ER membrane (Hiller et al., 1996; Werner et al., 1996; Wiertz et al., 1996; Plemper et al., 1997). Our current knowledge about the discovery of misfolded or unassembled proteins and their retention in the ER is still very limited. However, it is clear that the control mechanism works by structural rather than functional 2310 ER protein quality control criteria. Mutations in CFTR and a-1-antitrypsin, for example, which do not perturb the biological activity of the proteins per se, lead to ER retention and elimination of the mutant molecules, thus causing disease (Jensen et al., 1995; Ward et al., 1995; Qu et al., 1996). To date, a chaperone-mediated retention mechanism has been described only for mutant glycoproteins in mammalian systems. As proteins become translocated across the ER membrane, core oligosaccharides of the Glc 3 Man 9 - GlcNAc 2 structure are co-translationally attached to the side chains of asparagine residues within the Asn-X-Ser/ Thr consensus sequence. As folding progresses, two of the outermost glucose residues of the N-linked glycan are trimmed by the glucosidases I and II. In mammalian cells, exposure of the innermost glucose leads to the binding of the monoglucosylated protein to calnexin and calreticulin. If the glycoproteins contain cysteine residues, mixed disul de bonds are transiently formed by the thiol oxidoreductase ERp57, a member of the PDI family which speci cally interacts with calnexin, calreticulin and monoglucosylated glycoproteins, and functions as a disul de isomerase. When the remaining glucose residue is trimmed by glucosidase II, the complex dissociates, releasing a protein with the carbohydrate structure Man 9 GlcNAc 2. If the glycoprotein is not properly folded, the N-glycan is reglucosylated at the same position by a UDP-glucose glucosyltransferase (UGGT). This induces a new round of calnexin/calreticulin binding, which prevents the escape of the glycoprotein from the ER. If this cycle persists due to the inability of the glycoprotein to reach its nal native conformation, mannosidase I, a slowacting enzyme, cleaves the a1,2-linked mannose of the middle branch, generating a glycan with the structure Man 8 GlcNAc 2. Direct recognition of Man 8 GlcNAc 2 by a speci c lectin or attenuated release of the reglucosylated form (Glc 1 Man 8 GlcNAc 2 ) from calnexin are events which determine the delivery of malfolded glycoproteins to the elimination machinery (Cabral et al., 2001). In recent years, the yeast S.cerevisiae has set the pace in the eld of protein quality control in the ER (Figure 1). One of the model proteins used to study ER quality control in this organism is a mutated vacuolar enzyme, carboxypeptidase yscy-s255r, commonly known as CPY*. This misfolded protein is translocated into the ER lumen normally; it is fully glycosylated but is never transported to the vacuole. Instead, it is retained in the ER and degraded by the proteasome (Hiller et al., 1996; Knop et al., 1996a). It is interesting that ER quality control leads to degradation of misfolded proteins by the cytoplasmic proteasome machinery rather than by the vacuole (lysosome). A carbohydrate-based retention mechanism, which appears very similar to the mechanism described for mammalian cells, has also been identi ed for CPY*. Glucosidases I and II (Gls1, Gls2), and a-1,2 mannosidase (Mns1) are necessary for the disposal of CPY* (Knop et al., 1996b; Jakob et al., 1998). As opposed to the mammalian system, there is no known glucosyltransferase in yeast that could reglucosylate the carbohydrate chains. Thus, trimming by glucosidases I and II, and later by a-1,2 mannosidase, is currently believed to be the timer for folding or, if unsuccessful, for degradation. The recently discovered lectin-like yeast protein Htm1/Mnl1p, and its mouse homolog EDEM, is believed to bind Man 8 GlcNAc 2 and provides us with a link between recognition and targeting for degradation (Hosokawa et al., 2001; Jakob et al., 2001; Nakatsukasa et al., 2001) (Figure 1). Htm1p/ Mnl1p is also necessary for the ef cient degradation of other misfolded glycoproteins with differing topologies, such as Pdr5*, a mutated form of the ATP-binding cassette transporter Pdr5 (Plemper et al., 1998), and Stt3-7p, a mutant subunit of the OST complex (Jakob et al., 2001). On the other hand, degradation of Sec61-2p (Biederer et al., 1996), which is not N-glycosylated, is totally independent of Htm1p/Mnl1p, a nding that emphasizes the necessity of this lectin speci cally for the recognition of misfolded glycoproteins (Jakob et al., 2001). At present, the overall impact of this carbohydrate-based recognition on the quality control and degradation process as a whole is not clear. Likewise, our knowledge of the mechanism underlying the discovery of misfolded or unassembled non-glycoproteins is minimal. An obvious possibility is recognition of exposed hydrophobic patches by chaperone-like molecules. The major Hsp70 chaperone of the ER, BiP (Kar2p), is known to play a vital role in binding hydrophobic protein stretches to allow post-translational protein import into the ER and for protein folding and assembly within the ER (Johnson and van Waes, 1999; Haigh and Johnson, 2002). Its general function in protein quality control, therefore, seemed pretty straightforward. It is required for the degradation of soluble proteins such as mutant pro-a-factor and CPY* in yeast (Plemper et al., 1997; Brodsky et al., 1999). Surprisingly, Kar2p becomes dispensable when CPY* is anchored to the ER membrane. Basically, conversion of the same misfolded protein, CPY*, from a soluble form to a membrane-bound form abolishes the requirement for Kar2p (BiP) in ER degradation (C.Taxis and D.H.Wolf, manuscript submitted for publication). Degradation of other membrane proteins is also independent of Kar2p (Plemper et al., 1998). This indicates that Kar2p (BiP) and its DnaJ-like partners, Jem1p and Scj1p (Nishikawa et al., 2001), do not have a general function in the quality control process. Instead, Kar2p activity seems limited to soluble proteins (Figure 1). One function of Kar2p appears to reside in its ability to remain bound to misfolded proteins after their unsuccessful folding trials, essentially keeping them in soluble form. Scj1p and Jem1p may be necessary for triggering the release of Kar2p from such substrates in order for dislocation and degradation to take place. BiP and its partners may also play a role in the delivery of the soluble substrates to other, as yet unknown, components linking recognition to elimination (Nishikawa et al., 2001). BiP has also been implicated in acting as a seal for the protein translocation channel from the lumenal side (Johnson and van Waes, 1999; Haigh and Johnson, 2002). If BiP (Kar2p) has some function as a gatekeeper for the retrograde transport of malfolded proteins out of the ER, then this role would be limited to soluble proteins (C.Taxis and D.H.Wolf, manuscript submitted for publication). The requirement for Ca 2+ ions in the ER for degradation of soluble CPY* may be partly explained by their regulatory role in Kar2p activity (DuÈrr et al., 1998). PDI, an oxidoreductase involved in disul de bond formation in the ER (Regeimbal and Bardwell, 2002), is emerging as another component with multiple functions. While elimination of CPY* (Gillece et al., 1999) and unassembled 2311 Z.Kostova and D.H.Wolf Ig-m chains (Fagioli et al., 2001) requires the enzymatic activity of PDI, elimination of the non-glycosylated and cysteine-free mutant pro-a-factor requires binding of PDI without enzymatic activity, suggesting a chaperone-like role for PDI under these circumstances (Gillece et al., 1999) (Figure 1). PDI has also been described to function as a redox-driven chaperone in the unfolding of the A1 chain of cholera toxin in the ER lumen, prior to its transport to the cytoplasm. In this scenario, PDI binds and unfolds the substrate in its reduced state. The complex is then targeted to the ER membrane, where it binds to a protein at the lumenal side of the membrane. Oxidation of PDI by Ero1p releases the substrate, possibly directly into the retrotranslocon (Tsai and Rapoport, 2002). However, this redox-dependent mechanism has been questioned by Lumb and Bulleid's (2002) nding that PDI binding and release to two other substrates is not driven by the redox state of the protein. The necessity for Eps1p, a membranebound protein from the PDI family, in the elimination of a mutant form of the yeast membrane ATPase Pma1p (Wang and Chang, 1999), underlines the general necessity of this class of proteins as part of the ER protein quality control machinery. Calnexin represents yet another example of a multifunctional chaperone. In mammalian cells, it binds glycan moieties of proteins as expected from a lectin. In yeast, it is dispensable for CPY* degradation but it is involved in the elimination of the nonglycosylated mutant pro-a-factor (McCracken and Brodsky, 1996), suggesting additional non-lectin activity for this protein. The protein quality control machinery of the ER is also involved in the regulated degradation of ER-resident enzymes. The best-studied example in yeast is a key enzyme in the sterol biosynthetic pathway, HMGCoAreductase 2. Signals from the mevalonate pathway are presumed to lead to conformational changes which channel the protein into the ER quality control pathway (Hampton, 2002). Elimination of this enzyme requires all of the major components of the ER degradation machinery (Hampton et al., 1996; Hampton and Bhakta, 1997; Gardner et al., 2000; Bays et al., 2001a,b). Retrotranslocation It was initially believed that misfolded and orphan proteins of the ER were degraded by ER-resident proteinases and peptidases (Bonifacino and Klausner, 1994). However, the presence of unspeci c proteinases in the ER was hard to reconcile with its primary function in folding and assembly. Rather, delivery of misfolded proteins to the lysosome/vacuole, the compartment of the cell associated with degradation, seemed more likely. The discovery of the involvement of the cytoplasmic ubiquitin±proteasome system in the degradation of mutant ER membrane proteins was an unexpected surprise (Sommer and Jentsch, 1993; Jensen et al., 1995; Ward et al., 1995). The ndings, in S.cerevisiae, that the misfolded vacuolar peptidase CPY* (Hiller et al., 1996) and the mutant secretory protein pro-a-factor (Werner et al., 1996) were retained in the ER lumen and degraded in the cytoplasm by the ubiquitin±proteasome system, were crucial in establishing the concept of retrograde transport out of the ER. Genetic studies in yeast revealed a signi cantly retarded degradation of CPY* (Plemper et al., 1997), mutated proa-factor (Pilon et al., 1997) and a mutant polytopic membrane protein, Pdr5* (Plemper et al., 1998), in mutants defective in the Sec61 translocon. Co-immunoprecipitation studies indicated that Sec61b was associated with the major histocompatibility complex class I (MHC class I) heavy chains, with wild-type and mutant forms of the CFTR and with a mutant form of ribophorin I (RI332) during their dislocation from the ER (Kostova and Wolf, 2002). These studies support the notion that the Sec61 import channel may also be the export channel. It is very likely, though, that the retrotranslocation channel differs in its composition from the import channel. The Sec61 translocon is composed of three different subunits: Sec61a, Sec61b and Sec61g (Sec61p, Sbh1p and Sss1p in yeast). Studies on the retrotranslocon have identi ed a genetic interaction between Hrd3p and Sec61p (Plemper et al., 1999a; P.Deak and D.H.Wolf, unpublished
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