In general, the ERAD process can be divided in at least four stages: (i) recognition of the aberrant polypeptide, (ii) dislocation, (iii) release from the ER surface, and iv) degradation. However, it is becoming clear that rather than constituting a single defined pathway, ERAD can be considered as a collection of different pathways that may involve different players depending on the characteristics of the protein substrate in question.
Different mechanisms can contribute to the recognition of misfolded proteins in the ER. The same molecular chaperones that assist the folding of newly synthesized polypeptides have been often implicated in the process that prepares aberrant proteins for degradation. Indeed, chaperone interactions, aside from their value in facilitating protein folding may be important in maintaining proteins in soluble form and/or in targeting any terminally misfolded proteins to the next step of the ERAD pathway (dislocation).
The plant homologue of the immunoglobulin heavy chain binding protein (BiP) is a major ER chaperone of the Hsp70 protein family. Certain ERAD substrates have been found to bind extensively to BiP in the ER, their degradation being tightly associated with their release from the chaperone (Knittler et al. 1995; Skowronek et al. 1998; Chillaron et al. 2000; Molinari et al. 2002). Degradation of some, but not all, ERAD substrates has been found to be impaired in the presence of different BiP mutants (Plemper et al. 1997; Brodsky et al. 1999; Nishikawa et al. 2001), and aggregation of some misfolded proteins has been found to be exacerbated in yeast strains carrying mutated BiP (Nishikawa et al. 2001; Kabani et al. 2003). So, although BiP may play different roles in ERAD, one of them appears to be to maintain particular protein substrates in a soluble, retrotranslocation-competent state.
Different ER oxidoreductases including yeast Eps1p (Wang and Chang 2003), ERp57 (Antoniou et al. 2002), and protein disulfide isomerase (PDI) (Molinari et al. 2002; Tsai et al. 2001) have also been implicated in the disposal of ERAD substrates. Although extensive unfolding may not be strictly required for dislocation (Tirosh et al. 2002; Fiebiger et al. 2002), reduction of disulfide bonds has been shown to precede dislocation of IgM heavy chains (Fagioli et al. 2001) and ER oxidoreductases are obvious candidates as the catalyzers of reducing reactions required to prepare disulphide-bonded substrates for the membrane translocation step (Molinari et al. 2002). However, it should be noted that yeast PDI has been implicated in the disposal of a cysteine-free polypeptide, indicating that substrate reduction is not the only activity of PDI involved in quality control (Gillece et al. 1999). A physiological redox state is important not only to allow substrate reduction, but also for the general functioning of the ERAD machinery, since a cysteine-free protein is stabilized in the ER by treatments that affect intracellular redox potential or free thiol status (Tortorella et al. 1998). Finally, it is interesting to note that the degradation of certain membrane proteins has been found to require the action of cytosolic chaperones such as Hsp70 and Hsp104p, the latter being a yeast protein belonging to the AAA-ATPase superfamily (Hill and Cooper 2000; Zhang et al. 2001; Taxis et al. 2003). The activity of cytosolic chaperones appears to be specifically required for the disposal of large or tightly folded cytosolic domains of certain membrane proteins (Taxis et al. 2003).
Although interaction with chaperones is clearly important to prepare ERAD substrates for degradation, it is also becoming evident that other factors contribute to divert unfolded proteins from the biosynthetic to the degradative pathway. Within this context, the best characterized recognition mechanism within the ER is the one based on specific modifications of N-linked glycan chains. Many of the proteins that are inserted into the ER are modified by the addition of a core glycan of 14 saccharides (Fig. 1), which is transferred from a lipid carrier to asparagine residues within the sequon Asn-X-Ser/Thr, where X can be any amino acid but proline. The structure of the glycan initially transferred to the protein is the same in virtually all eukary-otes, and this high level of conservation likely reflects a conserved functional role. The large polar structures of core glycans can have a direct effect on protein stability and solubility, but also contribute to protein folding by mediating the interaction of nascent and newly synthesized glycoproteins with two ER chaperones, calnexin and calreticulin (see chapt. by Vitale and Denecke, this volume). While calnexin is a type I membrane protein, calreticulin is a soluble protein. Briefly, these chaperones are related to the legume lectin family and both of them interact with monoglucosylated oligosaccharides produced by the action of ER glucosidase I and glucosidase II (Fig. 1). Most importantly, monoglucosylated oligosaccharides are also produced by the action of UDP-glucose:glycoprotein glucosyltransferase (GT), a folding sensor that can selectively and iteratively add a glucose residue to misfolded (but not to folded) proteins, giving them further opportunities to interact with calnexin and calreticulin, and thus to complete structural maturation.
In several cases, it has been observed that inhibition of glucose trimming, and hence of calnexin/calreticulin binding, accelerates the degradation of misfolded proteins (de Virgilio et al. 1999; Wilson et al. 2000; Chung et al. 2000; Molinari et al. 2002; Mancini et al. 2003). Although this indicates that entry into the calnexin/calreticulin cycle can protect certain substrates from degradation, stabilizing ERAD substrates in their monoglucosylated form not always resulted in a prolonged half-life. While degradation of certain substrates was retarded (Molinari et al. 2002; Cabral et al. 2002; Oda et al. 2003),
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