Membrane-bound Ubx2 recruits Cdc48 to ubiquitin ligases and their substrates to ensure efficient ER-associated protein degradation

Endoplasmic reticulum (ER)-associated protein degradation (ERAD) is a quality control system that removes misfolded proteins from the ER. ERAD substrates are channelled from the ER via a proteinacious pore to the cytosolic ubiquitin-proteasome system - a process involving dedicated ubiquitin ligases and the chaperone-like AAA ATPase Cdc48 (also known as p97). How the activities of these proteins are coupled remains unclear. Here we show that the UBX domain protein Ubx2 is an integral ER membrane protein that recruits Cdc48 to the ER. Moreover, Ubx2 mediates binding of Cdc48 to the ubiquitin ligases Hrd1 and Doa10, and to ERAD substrates. In addition, Ubx2 and Cdc48 interact with Der1 and Dfm1, yeast homologues of the putative dislocation pore protein Derlin-1 (refs 11-13). Lack of Ubx2 causes defects in ERAD that are exacerbated under stress conditions. These findings are consistent with a model in which Ubx2 coordinates the assembly of a highly efficient ERAD machinery at the ER membrane.     

Shp1 and Ubx2 are adaptors of Cdc48 involved in ubiquitin-dependent protein degradation

Known activities of the ubiquitin-selective AAA ATPase Cdc48 (p97) require one of the mutually exclusive cofactors Ufd1/Npl4 and Shp1 (p47). Whereas Ufd1/Npl4 recruits Cdc48 to ubiquitylated proteins destined for degradation by the 26S proteasome, the UBX domain protein p47 has so far been linked exclusively to nondegradative Cdc48 functions in membrane fusion processes. Here, we show that all seven UBX domain proteins of Saccharomyces cerevisiae bind to Cdc48, thus constituting an entire new family of Cdc48 cofactors. The two major yeast UBX domain proteins, Shp1 and Ubx2, possess a ubiquitin-binding UBA domain and interact with ubiquitylated proteins in vivo. Deltashp1 and Deltaubx2 strains display defects in the degradation of a ubiquitylated model substrate, are sensitive to various stress conditions and are genetically linked to the 26S proteasome. Our data suggest that Shp1 and Ubx2 are adaptors for Cdc48-dependent protein degradation through the ubiquitin/proteasome pathway.     

Dfm1 Forms Distinct Complexes with Cdc48 and the ER Ubiquitin Ligases and Is Required for ERAD

Proteins imported into the endoplasmic reticulum (ER) are scanned for their folding status. Those that do not reach their native conformation are degraded via the ubiquitin‐proteasome system. This process is called ER‐associated degradation (ERAD). Der1 is known to be one of the components required for efficient degradation of soluble ERAD substrates like CPY^* (mutated carboxypeptidase yscY). A homologue of Der1 exists, named Dfm1. No function of Dfm1 has been discovered, although a C‐terminally hemagglutinin (HA)₃‐tagged Dfm1 protein has been shown to interact with the ERAD machinery. In our studies, we found Dfm1‐HA₃ to be an ERAD substrate and therefore not suitable for functional studies of Dfm1 in ERAD. We found cellular, non‐tagged Dfm1 to be a stable protein. We identified Dfm1 to be part of complexes which contain the ERAD‐L ligase Hrd1/Der3 and Der1 as well as the ERAD‐C ligase Doa10. In addition, ERAD of Ste6^*‐HA₃ was strongly dependent on Dfm1. Interestingly, Dfm1 forms a complex with the AAA‐ATPase Cdc48 in a strain lacking the Cdc48 membrane‐recruiting component Ubx2. This complex does not contain the ubiquitin ligases Hrd1/Der3 and Doa10. The existence of such a complex might point to an additional function of Dfm1 independent from ERAD.     

Quality control: another player joins the ERAD cast

The quality control system known as ERAD removes misfolded proteins from the ER to the cytosol for degradation. The AAA ATPase Cdc48p and ubiquitin ligases play crucial roles; their relationship has been unclear, but recent work has shown that the membrane protein Ubx2p links their functions in yeast.     

Cdc48p is UBX-linked to ER ubiquitin ligases

Proteasome-mediated turnover of misfolded secretory and transmembrane proteins at the cytoplasmic face of the endoplasmic reticulum (ER) membrane is dependent on a AAA-ATPase complex formed by the ubiquitin-selective chaperone Cdc48p in Saccharomyces cerevisiae and mammals by the Cdc48p homologue p97. Two new papers reveal that the Ubx2 protein physically links ER-membrane-integrated ubiquitin ligases to Cdc48p, and that it is essential for degradation of substrates that are ubiquitylated at the cytoplasmic face of the ER.     

The Ubx2 and Ubx3 cofactors direct Cdc48 activity to proteolytic and nonproteolytic ubiquitin-dependent processes

Valosin-containing protein, VCP/p97 or Cdc48, is a eukaryotic ATPase involved in membrane fusion, protein transport, and protein degradation. We describe two proteins, Ubx2 and Ubx3, which interact with Cdc48 in fission yeast. Ubx3 is the ortholog of p47/Shp1, a previously described Cdc48 cofactor involved in membrane fusion, whereas Ubx2 is a novel protein. Cdc48 binds the UBX domains present in both Ubx2 and Ubx3, indicating that this domain is a general Cdc48-interacting module. Ubx2 and Ubx3 also interact with ubiquitin chains. Disruption of the ubx3(+)-gene causes both temperature and canavanine sensitivity and stabilizes some ubiquitin-protein conjugates including the CDK inhibitor Rum1, but not a model substrate of the ER-degradation pathway. Moreover the ubx3 null displays synthetic lethality with a pus1 null mutant, a multiubiquitin binding subunit of the 26S proteasome. In contrast, the ubx2 null mutant did not display any obvious protein-degradation phenotype. In conclusion Ubx3/p47 is not, as previously thought, only important for membrane fusion; it's also important for the specific degradation of a subset of cell proteins. Our genetic analyses revealed that Ubx3/p47 functionally parallels a substrate receptor of the 26S proteasome, Pus1/Rpn10, indicating that the Cdc48-Ubx3 complex is involved in delivering substrates to the 26S proteasome.     

Doa1 is a Cdc48 adapter that possesses a novel ubiquitin binding domain

Cdc48 (p97/VCP) is an AAA-ATPase molecular chaperone whose cellular functions are facilitated by its interaction with ubiquitin binding cofactors (e.g., Npl4-Ufd1 and Shp1). Several studies have shown that Saccharomyces cerevisiae Doa1 (Ufd3/Zzz4) and its mammalian homologue, PLAA, interact with Cdc48. However, the function of this interaction has not been determined, nor has a physiological link between these proteins been demonstrated. Herein, we demonstrate that Cdc48 interacts directly with the C-terminal PUL domain of Doa1. We find that Doa1 possesses a novel ubiquitin binding domain (we propose the name PFU domain, for PLAA family ubiquitin binding domain), which appears to be necessary for Doa1 function. Our data suggest that the PUL and PFU domains of Doa1 promote the formation of a Doa1-Cdc48-ubiquitin ternary complex, potentially allowing for the recruitment of ubiquitinated proteins to Cdc48. DOA1 and CDC48 mutations are epistatic, suggesting that their interaction is physiologically relevant. Lastly, we provide evidence of functional conservation within the PLAA family by showing that a human-yeast chimera binds to ubiquitin and complements doa1Delta phenotypes in yeast. Combined, our data suggest that Doa1 plays a physiological role as a ubiquitin binding cofactor of Cdc48 and that human PLAA may play an analogous role via its interaction with p97/VCP.     

Doa1 targets ubiquitinated substrates for mitochondria-associated degradation

Mitochondria-associated degradation (MAD) mediated by the Cdc48 complex and proteasome degrades ubiquitinated mitochondrial outer-membrane proteins. MAD is critical for mitochondrial proteostasis, but it remains poorly characterized. We identified several mitochondrial Cdc48 substrates and developed a genetic screen assay to uncover regulators of the Cdc48-dependent MAD pathway. Surprisingly, we identified Doa1, a substrate-processing factor of Cdc48 that inhibits the degradation of some Cdc48 substrates, as a critical mediator of the turnover of mitochondrial Cdc48 substrates. Deletion of DOA1 causes the accumulation and mislocalization of substrates on mitochondria. Profiling of Cdc48 cofactors shows that Doa1 and Cdc48^-Ufd1-Npl4 form a functional complex mediating MAD. Biochemically, Doa1 interacts with ubiquitinated substrates and facilitates substrate recruitment to the Cdc48^-Ufd1-Npl4 complex. Functionally, Doa1 is critical for cell survival under mitochondrial oxidative stress, but not ER stress, conditions. Collectively, our results demonstrate the essential role of the Doa1–Cdc48^-Ufd1-Npl4 complex in mitochondrial proteostasis and suggest that Doa1 plays dual roles on the Cdc48 complex.     

Lipid droplets go nuclear

Dislocation of polypeptides from the mitochondrial outer membrane by the p97/Cdc48–Ufd1–Npl4 adenosine triphosphatase complex is essential for mitochondria-associated degradation and Parkin-mediated mitophagy. In this issue, Wu et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201510098) identify Doa1 as a pivotal adaptor that recruits substrates to Cdc48 for processing.Eukaryotic cells use the ubiquitin proteasome system to eliminate misfolded proteins from diverse subcellular compartments to maintain protein homeostasis. Once polyubiquitinated, soluble proteins are readily targeted to the proteasome. However, the degradation of proteins in lipid bilayer or membrane-encircled organelles requires additional steps because the membranes render these substrates, at least in part, inaccessible to the ubiquitin proteasome system. Taking ER-associated degradation (ERAD) as an example, misfolded ER luminal proteins can only become ubiquitinated after they emerge from the ER lumen via a retrotranslocation process; the degradation of ubiquitinated substrates embedded in the membrane then requires their dislocation into the cytosol, a reaction mediated by a conserved ATPase named p97 in mammals or Cdc48 in Saccharomyces cerevisiae (Christianson and Ye, 2014). During this process, the hexameric barrel-like ATPase p97/Cdc48, assisted by an array of cofactors, uses the energy from ATP hydrolysis to extract polypeptides from the membranes for targeting to the proteasome. Besides ERAD, p97/Cdc48 is also involved in dislocating polypeptides from the mitochondrial outer membranes (MOMs) to facilitate mitochondria-associated degradation (MAD; Heo et al., 2010; Xu et al., 2011; Hemion et al., 2014). The MAD process can be used to eliminate aberrant proteins for regulation of mitochondria protein homeostasis or to degrade factors (e.g., mitofusin) controlling the turnover of damaged mitochondria by mitophagy (Tanaka et al., 2010). Intriguingly, p97 and the heterodimeric cofactor Ufd1-Npl4 accumulate on the surface of damaged mitochondria, and deficiency in each of these factors causes a defect in Parkin-mediated mitophagy (Kimura et al., 2013). These findings suggest a critical role of p97/Cdc48 in mitochondria homeostasis regulation, but how substrates are recruited to p97/Cdc48 in MAD is unclear. In this issue, Wu et al. identify Doa1 as a critical regulator of Cdc48-dependent MAD in Saccharomyces cerevisiae (Wu et al., 2016).The MAD pathway has been poorly characterized in budding yeast because of a lack of model substrates, so Wu et al. (2016) first measured the half-life of endogenously tagged MOM proteins to identify substrates suitable for mechanistic characterization of this process. The study revealed four short-lived proteins (Fzo1, Mdm34, Msp1, and Tom70) whose rapid turnover depends not only on the proteasome but also on Cdc48. Among these substrates, Tom70, a mitochondrial import receptor, was used to set up a transposon-based genetic screen. The screen, notwithstanding its limited genome coverage (∼15%), efficiently uncovered four insertion mutants with elevated Tom70 expression that were likely associated with defects in MAD because the affected genes encode a proteasome-associated deubiquitinase (Ubp6), a deubiquitinase-binding protein (Bro1), an E3 ubiquitin ligase (Rsp5), and the Cdc48 adaptor Doa1. Because Doa1 was the only factor required for efficient degradation of all four MAD substrates, the authors further characterized its function.DOA1 (also named UFD3) was initially reported in a genetic screen that searched for genes required for efficient degradation of a β-galactosidase fusion protein containing a ubiquitin moiety at the N terminus. The screen identified five mutants, namely, ufd1–5 (ubiquitin fusion degradation; Johnson et al., 1995). Subsequent studies established several of these UFD proteins as key Cdc48-binding proteins required for Cdc48-dependent degradation. These include a substrate-recruiting adaptor (Ufd1) and a substrate-processing cofactor (Ufd2; Koegl et al., 1999; Meyer et al., 2000; Böhm et al., 2011). The WD domain–containing Doa1 was also shown to bind Cdc48, but it competes with Ufd2 in binding to the C terminus of Cdc48, and therefore was proposed to antagonize Ufd2 functionally in Cdc48-mediated degradation (Rumpf and Jentsch, 2006). However, because DOA1 deficiency also causes a reduction in the level of endogenous ubiquitin, which could indirectly stabilize proteasome substrates (Johnson et al., 1995), whether Doa1 negatively regulates Cdc48-mediated degradation has been unclear. Strikingly, Wu et al. (2016) showed that in the case of MAD, reexpression of ubiquitin in DOA1 null cells did not restore degradation. Thus, Wu et al. (2016) for the first time reveal a role of Doa1 in Cdc48-dependent degradation that is unrelated to its function in ubiquitin homeostasis regulation. Doa1 contains an N-terminal WD domain that has a strong ubiquitin-binding activity (Pashkova et al., 2010), a weak ubiquitin-binding PFU domain (Fu et al., 2009), and a C-terminal Cdc48-binding PUL domain (Ghislain et al., 1996; Mullally et al., 2006; Zhao et al., 2009; Qiu et al., 2010). Wu et al. (2016) performed complementation experiments with a series of Doa1 truncation mutants and showed that the WD domain and the PUL domain of Doa1 are indispensable for MAD, whereas the PFU domain is only required for degradation of a subset of MAD substrates.In ERAD, Cdc48/p97 is known to interact with ubiquitinated substrates and extract them from the ER membranes (Ye et al., 2001). Cdc48–Doa1 may act similarly in MAD because an interaction between Cdc48 and MAD substrates was observed by coimmunoprecipitation and because deletion of DOA1 caused MAD substrates to accumulate on mitochondrial membranes. Furthermore, biochemical fractionation showed that in cells bearing a temperature-sensitive cdc48 allele or lacking DOA1, MAD substrates enriched in the mitochondrial fraction are highly ubiquitinated.Comprehensive analyses of other known Cdc48 cofactors showed that in addition to Doa1, the Ufd1–Npl4 complex is also required for degradation of Cdc48 substrates at the mitochondria. As Ufd1–Npl4 binds to Cdc48 via its N-terminal domain, whereas Doa1 interacts with the C-terminal tail of Cdc48, a multiprotein complex consisting of Cdc48, Ufd1, Npl4, and Doa1 could be detected by coimmunoprecipitation. Genetic studies showed that both Doa1 and Npl4 are required for substrate interaction with Cdc48 in MAD, suggesting that these factors may function as a substrate-recruiting cofactor. Interestingly, the interaction of Doa1 with ubiquitinated MAD substrates, while being mediated by its WD40 domain, is also dependent on Cdc48: in cdc48-3 mutant cells, Doa1 accumulates on the mitochondrial membranes and binds MAD substrates more efficiently, yet deletion of the Cdc48-interacting domain reduced the interaction of Doa1 with MAD substrates. These results suggest that Doa1 may facilitate substrate recruitment to Cdc48 only when it is bound to Cdc48; but upon Cdc48-mediated extraction, substrates are released from this complex (Fig. 1).Open in a separate windowFigure 1.Cdc48–Doa1–dependent degradation of MOM proteins. A MOM protein (red) is ubiquitinated by an E3 ubiquitin ligase. The ubiquitin chain is then recognized by the ubiquitin-binding WD domain of Doa1 (light green), which recruits the substrate to the Cdc48–Ufd1–Npl4 complex through an interaction between its PUL domain (gray) and the Cdc48 C terminus. Upon ATP hydrolysis, Cdc48–Ufd1–Npl4 extracts ubiquitinated substrate from the MOM and brings it to the proteasome for degradation.The function of Doa1 in targeting proteins for degradation by the proteasome appears specific to MAD as deletion of DOA1 either had no effect on degradation of nonmitochondrial substrates or, in the case of the ERAD substrate CPY*, the stabilizing effect of DOA1 deletion could be attributed to the deficiency in ubiquitin. Moreover, unlike Ufd2, DOA1 deficiency did not sensitize cells to ER stress triggered by deletion of the IRE1 component of the unfolded protein response. In contrast, the growth of cells under increased mitochondrial oxidative stress conditions, such as superoxide dismutase deficiency, was compromised by deletion of the DOA1 gene, and this phenotype could be rescued by wild-type Doa1, but not by Doa1 mutants lacking either the WD or the Cdc48-binding PUL domain.Overall, Wu et al. (2016) convincingly establish Doa1 as a key regulator of MAD in S. cerevisiae, but whether Doa1’s mammalian homologue phospholipase A2 activating protein is similarly involved in MAD as well as in Parkin-mediated mitophagy remains to be tested. Because Doa1 binds to Cdc48 at a site close to the hexameric ring formed by the second ATPase domain (D2; Mullally et al., 2006; Rumpf and Jentsch, 2006; Zhao et al., 2009; Qiu et al., 2010), these findings further suggest that Cdc48 and perhaps its mammalian homologue p97 might first engage substrate using the D2 domain, as proposed previously (DeLaBarre et al., 2006). Intriguingly, the N domain–binding cofactor Ufd1–Npl4 is also required for substrate interaction with Cdc48/p97 in MAD. It is unclear how substrate recruitment to Cdc48/p97 could simultaneously involve two spatially separated adaptors. One possibility is that Ufd1–Npl4 may indirectly promote substrate binding by allosterically activating Cdc48/p97 or Doa1. Alternatively, these adaptors may relay substrate for targeting to Cdc48. The key to distinguish between these models lies in better assays that would allow the mapping of direct interactions between Cdc48 and MAD substrates in the presence or absence of these adaptors. The precise signal for substrate recognition in Cdc48p–Doa1–mediated MAD also remains to be elucidated. Despite these unresolved issues, the newly identified substrates and the demonstration that Doa1 is the MAD adaptor for Cdc48 by Wu et al. (2016) should provide a new handle to advance our understanding of this important yet poorly studied pathway.     

Ubiquitylation in ERAD: Reversing to Go Forward?

Proteins are co-translationally inserted into the endoplasmic reticulum (ER) where they undergo maturation. Homeostasis in the ER requires a highly sensitive and selective means of quality control. This occurs through ER-associated degradation (ERAD).This complex ubiquitin-proteasome–mediated process involves ubiquitin conjugating enzymes (E2) and ubiquitin ligases (E3),lumenal and cytosolic chaperones, and other proteins, including the AAA ATPase p97 (VCP; Cdc48 in yeast). Probing of processes involving proteasomal degradation has generally depended on proteasome inhibitors or knockdown of specific E2s or E3s. In this issue of PLoS Biology, Ernst et al. demonstrate the utility of expressing the catalytic domain of a viral deubiquitylating enzyme to probe the ubiquitin system. Convincing evidence is provided that deubiquitylation is integral to dislocation of ERAD substrates from the ER membrane. The implications of this work for understanding ERAD and the potential of expressing deubiquitylating enzyme domains for studying ubiquitin-mediated processes are discussed.     

Direct binding of ubiquitin conjugates by the mammalian p97 adaptor complexes,p47 and Ufd1-Npl4

The multiple functions of the p97/Cdc48p ATPase can be explained largely by adaptors that link its activity to different cellular pathways, but how these adaptors recognize different substrates is unclear. Here we present evidence that the mammalian adaptors, p47 and Ufd1-Npl4, both bind ubiquitin conjugates directly and so link p97 to ubiquitylated substrates. In the case of Ufd1-Npl4, which is involved in endoplasmic reticulum (ER)-associated degradation and nuclear envelope reassembly, binding to ubiquitin is mediated through a putative zinc finger in Npl4. This novel domain (NZF) is conserved in metazoa and is both present and functional in other proteins. In the case of p47, which is involved in the reassembly of the ER, the nuclear envelope and the Golgi apparatus, binding is mediated by a UBA domain. Unlike Ufd1-Npl4, it binds ubiquitin only when complexed with p97, and binds mono- rather than polyubiquitin conjugates. The UBA domain is required for the function of p47 in mitotic Golgi reassembly. Together, these data suggest that ubiquitin recognition is a common feature of p97-mediated reactions.     

Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins

Many misfolded endoplasmic reticulum (ER) proteins are eliminated by ERAD, a process in which substrates are polyubiquitylated and moved into the cytosol for proteasomal degradation. We have identified in S. cerevisiae distinct ubiquitin-ligase complexes that define different ERAD pathways. Proteins with misfolded ER-luminal domains use the ERAD-L pathway, in which the Hrd1p/Hrd3p ligase forms a near stoichiometric membrane core complex by binding to Der1p via the linker protein Usa1p. This core complex associates through Hrd3p with Yos9p, a substrate recognition protein in the ER lumen. Substrates with misfolded intramembrane domains define a pathway (ERAD-M) that differs from ERAD-L by being independent of Usa1p and Der1p. Membrane proteins with misfolded cytosolic domains use the ERAD-C pathway and are directly targeted to the Doa10p ubiquitin ligase. All three pathways converge at the Cdc48p ATPase complex. These results lead to a unifying concept for ERAD that may also apply to mammalian cells.     

Cdc48p(Npl4p/Ufd1p) binds and segregates membrane-anchored/tethered complexes via a polyubiquitin signal present on the anchors

Cdc48p is an abundant and conserved member of the AAA ATPase family of molecular chaperones. Cdc48p performs ubiquitin-selective functions, which are mediated by numerous ubiquitin binding adaptors, including the Npl4p-Ufd1p complex. Previous studies suggest that Cdc48p-containing complexes carry out many biochemical activities, including ubiquitination, deubiquitination, protein complex segregation, and targeting of ubiquitinated substrates to the proteasome. The molecular mechanisms by which Cdc48p-containing complexes participate in these processes remain poorly defined. We show here by using physiologically relevant Cdc48p substrates (i.e., endoplasmic membrane-associated/tethered dimers of Mga2p and Spt23p) and in vitro systems with purified proteins that Cdc48p(Npl4p/Ufd1p) binds to and promotes segregation of the tethered proteins via a polyubiquitin signal present on the membrane-bound proteins. Mobilization does not involve retrotranslocation of the associated anchors. These results provide biochemical evidence that Cdc48p(Npl4p/Ufd1p) functions as a polyubiquitin-selective segregase and that a polyubiquitin-Cdc48p pathway modulates protein interactions at cell membranes.     

Molecular determinants of the interaction between Doa1 and Hse1 involved in endosomal sorting

Yeast Doa1/Ufd3 is an adaptor protein for Cdc48 (p97 in mammal), an AAA type ATPase associated with endoplasmic reticulum-associated protein degradation pathway and endosomal sorting into multivesicular bodies. Doa1 functions in the endosomal sorting by its association with Hse1, a component of endosomal sorting complex required for transport (ESCRT) system. The association of Doa1 with Hse1 was previously reported to be mediated between PFU domain of Doa1 and SH3 of Hse1. However, it remains unclear which residues are specifically involved in the interaction. Here we report that Doa1/PFU interacts with Hse1/SH3 with a moderate affinity of 5 μM. Asn-438 of Doa1/PFU and Trp-254 of Hse1/SH3 are found to be critical in the interaction while Phe-434, implicated in ubiquitin binding via a hydrophobic interaction, is not. Small-angle X-ray scattering measurements combined with molecular docking and biochemical analysis yield the solution structure of the Doa1/PFU:Hse1/SH3 complex. Taken together, our results suggest that hydrogen bonding is a major determinant in the interaction of Doa1/PFU with Hse1/SH3.     

Complex of Fas-associated Factor 1 (FAF1) with Valosin-containing Protein (VCP)-Npl4-Ufd1 and Polyubiquitinated Proteins Promotes Endoplasmic Reticulum-associated Degradation (ERAD)

Fas-associated factor 1 (FAF1) is a ubiquitin receptor containing multiple ubiquitin-related domains including ubiquitin-associated (UBA), ubiquitin-like (UBL) 1, UBL2, and ubiquitin regulatory X (UBX). We previously showed that N-terminal UBA domain recognizes Lys⁴⁸-ubiquitin linkage to recruit polyubiquitinated proteins and that a C-terminal UBX domain interacts with valosin-containing protein (VCP). This study shows that FAF1 interacts only with VCP complexed with Npl4-Ufd1 heterodimer, a requirement for the recruitment of polyubiquitinated proteins to UBA domain. Intriguingly, VCP association to C-terminal UBX domain regulates ubiquitin binding to N-terminal UBA domain without direct interaction between UBA and UBX domains. These interactions are well characterized by structural and biochemical analysis. VCP-Npl4-Ufd1 complex is known as the machinery required for endoplasmic reticulum-associated degradation. We demonstrate here that FAF1 binds to VCP-Npl4-Ufd1 complex via UBX domain and polyubiquitinated proteins via UBA domain to promote endoplasmic reticulum-associated degradation.     

The Hrd1p ligase complex forms a linchpin between ER-lumenal substrate selection and Cdc48p recruitment

Misfolded proteins of the endoplasmic reticulum (ER) are targeted to the cytoplasm for proteasomal degradation. Key components of this process are ER membrane-bound ubiquitin ligases. These ligases associate with the cytoplasmic AAA-ATPase Cdc48p/p97, which is thought to support the release of malfolded proteins from the ER. Here, we characterize a yeast protein complex containing the ubiquitin ligase Hrd1p and the ER membrane proteins Hrd3p and Der1p. Hrd3p binds malfolded proteins in the ER lumen enabling their delivery to downstream components. Therefore, we propose that Hrd3p acts as a substrate recruitment factor for the Hrd1p ligase complex. Hrd3p function is also required for the association of Cdc48p with Hrd1p. Moreover, our data demonstrate that recruitment of Cdc48p depends on substrate processing by the Hrd1p ligase complex. Thus, the Hrd1p ligase complex unites substrate selection in the ER lumen and polyubiquitination in the cytoplasm and links these processes to the release of ER proteins via the Cdc48p complex.     

213 Structures and interactions of proteins involved in ER-associated protein degradation

Proteins are translocated into the endoplasmic reticulum (ER) of cells in an unfolded state, and acquire their native conformation in the ER lumen after signal peptide cleavage. ER-associated degradation (ERAD) of folding-incompetent protein chains is mediated by the protein complexes residing in the ER membrane. We study the architecture and function of one of these, the HRD complex assembled around the E3 ubiquitin ligase Hrd1. The recognition of ERAD substrates is linked to the maturation of their carbohydrate structures. The HRD complex-associated lectin Yos9 is involved in ERAD substrate recognition by binding carbohydrates through its mannose-6-phosphate receptor homology (MRH) domain. We have determined the crystal structure of a central domain of Yos9, adjacent to the MRH domain, which was previously annotated as interaction region with the HRD subunit Hrd3 (Hanna et al., 2012). We find that this domain does not support Hrd3 association which we map to the N-terminal half of Yos9 instead. In contrast, the domain has a function in Yos9 dimerization as seen in the crystal structure, in various solution experiments and as supported by mutagenesis of dimer interface residues. The dimerization of the ER-luminal Yos9, in conjunction with studies of the cytosolic domain of the HRD component Usa1 (Horn et al., 2009) and other biochemical data thus supports a model of a HRD complex that exists and functions as a dimer or a higher multimer. The delivery of ubiquitinated ERAD substrates to the proteasome is mediated by the cytosolic AAA ATPase Cdc48 (p97 in mammalian cells). The p97 (VCP) serves a wide variety of cellular functions in addition to its role in ERAD, including organelle membrane fusion, mitosis, DNA repair, and apoptosis. These different functions are linked to the binding of adaptor proteins to p97, many of which contain ubiquitin regulatory X (UBX) domains. One of these adaptors, ASPL (alveolar soft part sarcoma locus), uses a substantially extended UBX domain for binding to the N domain of p97 where a lariat-like, mostly α-helical extension wraps around one subunit of p97. By this binding ASPL triggers the dissociation of functional p97 hexamers leading to partial inactivation of the AAA ATPase. To the best of our knowledge, this is the first time that the structural basis for adaptor protein-induced inactivation by hexamer dissociation of p97 and, indeed, any AAA ATPase has been demonstrated. This observation has far reaching implications for AAA ATPase-regulated processes.