Cleaning up in the endoplasmic reticulum: ubiquitin in charge

Abstract

The eukaryotic endoplasmic reticulum (ER) maintains protein homeostasis by eliminating unwanted proteins through the evolutionarily conserved ER-associated degradation (ERAD) pathway. During ERAD, maturation-defective and surplus polypeptides are evicted from the ER lumen and/or lipid bilayer through the process of retrotranslocation and ultimately degraded by the proteasome. An integral facet of the ERAD mechanism is the ubiquitin system, composed of the ubiquitin modifier and the factors for assembling, processing and binding ubiquitin chains on conjugated substrates. Beyond simply marking polypeptides for degradation, the ubiquitin system is functionally intertwined with retrotranslocation machinery to transport polypeptides across the ER membrane.

Figure 1

Distinct modes of retrotranslocation. Retrotranslocation can occur in principle via the following modes. (1) A luminal substrate must first be inserted into the ER membrane in a process that is probably facilitated by a membrane-bound retrotranslocon component. Once a portion of the substrate has been exposed to the cytosol, the ubiquitination machinery can access it to facilitate its ubiquitination and subsequent dislocation from the membrane. Retrotranslocation of membrane proteins may proceed via several routes. Ub, ubiquitin. (2) Type I membrane proteins containing charged residues in their TM domain might be completely translocated into the ER lumen and then retrotranslocated similarly to a luminal substrate. (3) Some membrane proteins may first be processed by an intramembranous protease to release its luminal domain, which is then retrotranslocated. (4) Some membrane proteins may translocate their ER-luminal domain out of the ER first via a proteinaceous channel, which is then ubiquitinated and pulled out of the membrane. The TM domain may enter the channel via a lateral gate before being dislocated from the membrane. (5) Some membrane proteins may be ubiquitinated in their cytosolic domain and then pulled into a proteinaceous channel before release from the ER membrane.

Figure 2

Modular organization of the mammalian ERAD system. A large number of ERAD factors are organized into several modules to mediate retrotranslocation. In general, proteins in the same module tend to form stable interactions, whereas factors from different modules bind each other in a dynamic manner, although there are exceptions. For instance, a large, stable membrane complex consisting of an E3 ligase and other factors is likely to mediate the retrotranslocation initiation and the ubiquitination modules. Within a given module, factors can also form parallel pathways to transport distinct classes of the substrates. For an ERAD-L mechanism, the module for substrate recognition and recruitment distinguishes misfolded proteins from folded ones in the lumen and then selectively targets them for insertion into the ER membranes. Subsequent ubiquitin conjugation is mediated by ubiquitination modules. This is followed by extraction from the ER membrane via the AAA+ ATPase p97/VCP complex. Dislocated products are shuttled to the proteasome, which may have been brought near to the retrotranslocation site through interaction with the ER membranes. The yeast ERAD system has a similar modular organization with fewer participating components. The retrotranslocation and degradation of ERAD-M and ERAD-C substrates do not seem to require the recognition and initiation modules in the ER. The dashed lines indicate the flow of the substrates.

(1) Polytopic ERAD E3s can form functional homo-oligomers to enhance ubiquitination activity71,82. Oligomerization of gp78 increases polyubiquitination efficiency, probably by promoting chain preassembly on the active site of the E2 Ube2g2, which can be transferred en bloc to a substrate90. (2) Unlike many other E3s, some ERAD E3s recruit cognate E2s through high-affinity interactions. The yeast Hrd1p interactor Cue1p contains a high-affinity binding region for the E2 Ubc7p (U7BR) that is required for efficient ubiquitination and degradation of Hrd1 substrates^,. Although there is no bona fide Cue1p homolog, two mammalian proteins, gp78 and AUP1, contain a similar Ube2g2-binding region (G2BR). Both U7BR and G2BR domains bind the ‘back side’ of the corresponding E2s, inducing allosteric changes in E2 that increase ubiquitin loading and transfer^,,. Stable E3-E2 interactions, when acting in the context of an E3 homo-oligomer, may enhance ubiquitination efficiency either by forming preassembled ubiquitin chains or by simultaneously positioning multiple E2s in proximity to substrates. (3) The G2BR also acts in conjunction with ubiquitin-binding domains (UBDs) to further improve chain-building efficiency. Both Cue1p and gp78 contain ubiquitin-binding CUE domains that promote chain elongation^–. (4) Functional cooperation among ubiquitinating enzymes (E2s or E3s) or between E3s and molecular chaperones may be another mechanism that promotes ubiquitination. Whereas one E2 (for example, Ubc6p) may be dedicated solely to chain initiation, another (for example, Ubc7p) may serve to elongate those chains. Likewise, one E3 (for example, RNF5) can initiate ubiquitin-chain formation, whereas another E3 (for example, gp78) elongates the chains, functioning similarly to an E4 (refs. –130). (5) Doa10p also associates with the cytosolic chaperone Hsp70, which promotes polyubiquitination, probably by increasing the substrate dwell time on Doa10p. Similar functional interplays between an E3 and a chaperone have also been demonstrated in mammalian ERAD.

The ERAD-L mechanism shares a striking resemblance to a peroxisome protein–import pathway and the symbiont-specific ERAD-like machinery (SELMA). In yeast, ERAD-L requires a membrane complex comprising the substrate adaptor protein Hrd3p, Der1p and the ubiquitin ligase Hrd1p. The substrates are moved across the ER membrane via this large membrane complex. On the cytosolic face, an adaptor, Ubx2p, recruits the Cdc48p–Ufd1p–Npl4p complex to the ER membrane, which extracts the substrate from the membrane. The import of peroxisome proteins bearing a C-terminal peroxisomal targeting signal 1 (PTS1) begins with the recognition of cargo in the cytosol by a receptor protein, Pex5. Like Hrd3p, Pex5 uses several tetratricopeptide repeats (TPRs) to bind substrates. The Pex5–cargo complex is recruited to the membrane by a docking complex (Pex13/14) that also contains several RING-finger E3 ligases, Pex2, Pex10 and Pex12 (Pex2/10/12). Pex5 is believed to form a pore after integration into the membrane, and the release of substrate cargos into the peroxisome lumen is coupled to the ubiquitination and subsequent extraction of Pex5 from the membrane. Cytosolic AAA+ ATPase Pex1 and Pex6 (Pex1/6) mediate the dislocation of Pex5 from the membrane via a process analogous to the extraction of ubiquitinated ERAD substrates. The SELMA is a protein-translocation system located in the second-outermost membrane of certain plastids found in some alga and human parasites. Proteins in the stroma of these plastids need to traverse four layers of membrane. After Sec61-dependent transport into the ER lumen, they use the SELMA to reach the periplastidal compartment (PPC) for further translocation into the stroma. The system appears to be adapted from the ERAD-L machinery, because it uses two Der1-like proteins (sDer1–1 and sDer1–2), a membrane-bound E3 ligase (ptE3P) and a Cdc48-like complex (sCdc48–Ufd1) to move polypeptides across this membrane.