Misfolded soluble secretory and membrane proteins, as well as the unassembled subunits of multimeric proteins, are often degraded within an hour or two after their synthesis in the rough ER. Initially, it was thought that proteolytic enzymes within the ER lumen catalyzed degradation of mis folded or unassembled polypeptides, but such proteases were never found. More recent studies have shown that misfolded secretory proteins are recognized by specific ER membrane proteins and are targeted for transport from the ER lumen into the cytosol by a process known as dislocation.
The dislocation and degradation of misfolded proteins depends on a set of proteins located in the ER membrane and in the cytosol that perform three basic functions. The first function is recognition of misfolded proteins. One mechanism for recognition involves the trimming of N linked carbohydrate chains by mannosidases located in the ER (Figure 1). Trimmed glycans with the structure Man5–6(GlcNAc)2 are recognized by a protein known as OS-9, which targets the trimmed glycoprotein for dislocation. Both EDEM and OS-9 target the trimmed glycoprotein for dislocation. It is not known precisely how the ER α-mannosidases distinguish proteins that cannot fold properly, and are thus legitimate substrates for the dislocation process, from nor mal proteins that have transient partially folded states as they acquire their fully folded conformation. One possibility is that the ER α-mannosidases act slowly, so that only those glycoproteins that remain misfolded in the ER lumen for a sufficiently long time are trimmed and therefore targeted for degradation. Luminal proteins that lack oligosaccharides al together can also be targeted for degradation, indicating that other processes for the recognition of unfolded proteins must also exist. Those other mechanisms cannot involve trimming of N-linked oligosaccharides because misfolded membrane proteins that lack N-linked oligosaccharides altogether can nevertheless be targeted for degradation.
Fig1. Modifications of N-linked oligosaccharides are used to monitor folding and for quality control. After three glucose residues are removed from N-linked oligosaccharides in the ER, a single glucose can be re-added by a glucosyltransferase to form Glc1Man9(GlcNAc)2. This modified N-linked oligosaccharide binds the lectins calnexin (CNX) and calreticulin (CRT) for retention in the ER and engagement of folding chaperones. Proteins that cannot fold properly, and are therefore retained in the ER for longer times, undergo mannose trimming by ER α-mannosidases to form Man5–6(GlcNAc)2, which is recognized by OS-9. Recognition by OS-9 leads to dislocation of the misfolded protein out of the ER, ubiquitinylation, and degradation by proteasomes.
The second function required for dislocation of misfolded proteins is the transport of the marked proteins from the lumen of the ER across the ER membrane to the cytosol. A complex of at least four integral membrane proteins, known as the ERAD (ER-associated degradation) complex, enables dislocation of misfolded proteins across the ER membrane. The mechanism by which misfolded proteins traverse the ER membrane is not yet known, but there is no evidence that the ERAD complex forms a protein channel for dislocation, and it may be that dislocation involves powerful pulling and unfolding protein machines in the cytosol that drag misfolded proteins directly through the lipid bilayer.
Finally, as segments of a dislocated polypeptide are exposed to the cytosol, they encounter cytosolic enzymes that effect their degradation. One of these enzymes is p97, a member of a protein family, known as the AAA ATPase family, that couples the energy of ATP hydrolysis to disassembly of protein complexes. In dislocation of polypeptides from the ER, hydrolysis of ATP by p97 may provide the driving force to pull misfolded proteins from the ER membrane into the cytosol. As the misfolded proteins enter the cytosol, specific ubiquitin ligase enzymes that are components of the ERAD complex add ubiquitin residues to the dislocated peptides. Like the action of p97, the ubiquitinylation reaction is coupled to ATP hydrolysis; this release of energy may also contribute to trapping the proteins in the cytosol. The resulting polyubiquitinylated polypeptides, now fully in the cytosol, are removed from the cell altogether by degradation in proteasomes.
