A stress-responsive system for mitochondrial protein degradation

We show that Ydr049 (renamed VCP/Cdc48-associated mitochondrial stress-responsive--Vms1), a member of an unstudied pan-eukaryotic protein family, translocates from the cytosol to mitochondria upon mitochondrial stress. Cells lacking Vms1 show progressive mitochondrial failure, hypersensitivity to oxidative stress, and decreased chronological life span. Both yeast and mammalian Vms1 stably interact with Cdc48/VCP/p97, a component of the ubiquitin/proteasome system with a well-defined role in endoplasmic reticulum-associated protein degradation (ERAD), wherein misfolded ER proteins are degraded in the cytosol. We show that oxidative stress triggers mitochondrial localization of Cdc48 and this is dependent on Vms1. When this system is impaired by mutation of Vms1, ubiquitin-dependent mitochondrial protein degradation, mitochondrial respiratory function, and cell viability are compromised. We demonstrate that Vms1 is a required component of an evolutionarily conserved system for mitochondrial protein degradation, which is necessary to maintain mitochondrial, cellular, and organismal viability.     

Cellular functions of Ufd2 and Ufd3 in proteasomal protein degradation depend on Cdc48 binding

The chaperone-related AAA ATPase Cdc48 (p97/VCP in higher eukaryotes) segregates ubiquitylated proteins for subsequent degradation by the 26S proteasome or for nonproteolytic fates. The specific outcome of Cdc48 activity is controlled by the evolutionary conserved cofactors Ufd2 and Ufd3, which antagonistically regulate the substrates' ubiquitylation states. In contrast to the interaction of Ufd3 and Cdc48, the interaction between the ubiquitin chain elongating enzyme Ufd2 and Cdc48 has not been precisely mapped. Consequently, it is still unknown whether physiological functions of Ufd2 in fact require Cdc48 binding. Here, we show that Ufd2 binds to the C-terminal tail of Cdc48, unlike the human Ufd2 homologue E4B, which interacts with the N domain of p97. The binding sites for Ufd2 and Ufd3 on Cdc48 overlap and depend critically on the conserved residue Y834 but are not identical. Saccharomyces cerevisiae cdc48 mutants altered in residue Y834 or lacking the C-terminal tail are viable and exhibit normal growth. Importantly, however, loss of Ufd2 and Ufd3 binding in these mutants phenocopies defects of Δufd2 and Δufd3 mutants in the ubiquitin fusion degradation (UFD) and Ole1 fatty acid desaturase activation (OLE) pathways. These results indicate that key cellular functions of Ufd2 and Ufd3 in proteasomal protein degradation require their interaction with Cdc48.     

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.     

Functional division of substrate processing cofactors of the ubiquitin-selective Cdc48 chaperone

Ubiquitin-dependent protein degradation usually involves escort factors that target ubiquitylated substrates to the proteasome. A central element in a major escort pathway is Cdc48, a chaperone-like AAA ATPase that collects ubiquitylated substrates via alternative substrate-recruiting cofactors. Cdc48 also associates with Ufd2, an E4 multiubiquitylation enzyme that adds further ubiquitin moieties to preformed ubiquitin conjugates to promote degradation. Here, we show that E4 can be counteracted in vivo by two distinct mechanisms. First, Ufd3, a WD40 repeat protein, directly competes with Ufd2, because both factors utilize the same docking site on Cdc48. Second, Cdc48 also binds Otu1, a deubiquitylation enzyme, which disassembles multiubiquitin chains. Notably, Cdc48 can bind Otu1 and Ufd3 simultaneously, making a cooperation of both inhibitory mechanisms possible. We propose that the balance between the distinct substrate-processing cofactors may determine whether a substrate is multiubiquitylated and routed to the proteasome for degradation or deubiquitylated and/or released for other purposes.     

Mitochondrial quality control by the ubiquitin-proteasome system

Mitochondria perform multiple functions critical to the maintenance of cellular homoeostasis and their dysfunction leads to disease. Several lines of evidence suggest the presence of a MAD (mitochondria-associated degradation) pathway that regulates mitochondrial protein quality control. Internal mitochondrial proteins may be retrotranslocated to the OMM (outer mitochondrial membrane), multiple E3 ubiquitin ligases reside at the OMM and inhibition of the proteasome causes accumulation of ubiquitinated proteins at the OMM. Reminiscent of ERAD [ER (endoplasmic reticulum)-associated degradation], Cdc48 (cell division cycle 42)/p97 is recruited to stressed mitochondria, extracts ubiquitinated proteins from the OMM and presents ubiquitinated proteins to the proteasome for degradation. Recent research has provided mechanistic insights into the interaction of the UPS (ubiquitin-proteasome system) with the OMM. In yeast, Vms1 [VCP (valosin-containing protein) (p97)/Cdc48-associated mitochondrial-stress-responsive 1] protein recruits Cdc48/p97 to the OMM. In mammalian systems, the E3 ubiquitin ligase parkin regulates the recruitment of Cdc48/p97 to mitochondria, subsequent mitochondrial protein degradation and mitochondrial autophagy. Disruption of the Vms1 or parkin systems results in the hyper-accumulation of ubiquitinated proteins at mitochondria and subsequent mitochondrial dysfunction. The emerging MAD pathway is important for the maintenance of cellular and therefore organismal viability.     

Cdc48p/p97-mediated regulation of mitochondrial morphology is Vms1p-independent

Cdc48p/p97 is a cytosolic essential AAA chaperone, which regulates multiple cellular reactions in a ubiquitin-dependent manner. We have recently shown that Cdc48p exhibits positively cooperative ATPase activity and loss of the positive cooperativity results in yeast cell death. Here we show that loss of the positive cooperativity of the yeast Cdc48p ATPase activity led to severe mitochondrial aggregation. The actin cytoskeleton and distribution of the ER-mitochondria tethering complex (ERMES) were eliminated from the cause of the mitochondrial aggregation. Instead, a mitochondrial outer membrane protein Fzo1p, which is required for mitochondrial fusion, and components of ERMES, which is involved in mitochondrial morphology, were remarkably stabilized in the Cdc48p mutants. In the last couple of years, it was shown that Vms1p functions as a cofactor of Cdc48p for the function of protein degradation of mitochondrial outer membrane proteins. Nevertheless, we found that Vms1p was not involved in the Cdc48p-dependent mitochondrial aggregation and loss of Vms1p did not significantly affect degradation rates of proteins anchored to the mitochondrial outer membrane. These results suggest that Cdc48p controls mitochondrial morphology by regulating turnover of proteins involved in mitochondrial morphology in a Vms1p-independent manner.     

A Cdc48p-associated factor modulates endoplasmic reticulum-associated degradation, cell stress, and ubiquitinated protein homeostasis

The hexameric AAA-ATPase, Cdc48p, catalyzes an array of cellular activities, including endoplasmic reticulum (ER)-associated degradation (ERAD), ER/Golgi membrane dynamics, and DNA replication. Accumulating data suggest that unique Cdc48p partners, such as Npl4p-Ufd1p and Ubx1p/Shp1p (p47 in vertebrates), target Cdc48p for these diverse functions. Other Cdc48p-associated proteins have been identified, but the interplay among these factors and their activities is largely cryptic. We now report on a previously uncharacterized Cdc48p-associated protein, Ydr049p, also known as Vms1p, which binds Cdc48p at both the ER membrane and in the cytosol under non-stressed conditions. Loss of YDR049 modestly slows the degradation of the cystic fibrosis transmembrane conductance regulator but does not impede substrate ubiquitination, suggesting that Ydr049p acts at a postubiquitination step in the ERAD pathway. Consistent with Ydr049p playing a role in Cdc48p substrate release, ydr049 mutant cells accumulate Cdc48p-bound ubiquitinated proteins at the ER membrane. Moreover, YDR049 interacts with genes encoding select UBX (ubiquitin regulatory X) and UFD (ubiquitin fusion degradation) proteins, which are Cdc48p partners. Exacerbated growth defects are apparent in some of the mutant combinations, and synergistic effects on the degradation of cystic fibrosis transmembrane conductance regulator and CPY*, which is a soluble ERAD substrate, are evident in specific ydr049-ufd and -ubx mutants. These data suggest that Ydr049p acts in parallel with Cdc48p partners to modulate ERAD and other cellular activities.     

Analysis of intracellular distribution and apoptosis involvement of the Ufd1l gene product by over-expression studies

UFD1L is the human homologue of the yeast ubiquitin fusion degradation 1 (Ufd1) gene and maps on chromosome 22q11.2 in the typically deleted region (TDR) for DiGeorge/velocardiofacial syndromes (DGS/VCFS). In yeast, Ufd1 protein is involved in a degradation pathway for ubiquitin fused products (UFD pathway). Several studies have demonstrated that Ufd1 is a component of the Cdc48-Ufd1-Npl4 multiprotein complex which is active in the recognition of several polyubiquitin-tagged proteins and facilitates their presentation to the 26S proteasome for protein degradation or even more specific processing. The multiprotein complex Cdc48-Ufd-Npl4 is also active in mammalian cells. The biochemical role of UFD1L protein in human cells is unknown, even though the interaction between UFD1L and NPL4 proteins has been maintained. In order to clarify this issue, we examined the intracellular distribution of the protein in different mammalian cells and studied its involvement in the Fas and ceramide factors-mediated apoptotic pathways. We established that in mammalian cells, Ufd1l is localized around the nucleus and that it does not interfere with Fas-and ceramide-mediated apoptosis.     

Ufd1 exhibits the AAA-ATPase fold with two distinct ubiquitin interaction sites

Ufd1 mediates ubiquitin fusion degradation by association with Npl4 and Cdc48/p97. The Ufd1-ubiquitin interaction is essential for transfer of substrates to the proteasome. However, the mechanism and specificity of ubiquitin recognition by Ufd1 are poorly understood due to the lack of detailed structural information. Here, we present the solution structure of yeast Ufd1 N domain and show that it has two distinct binding sites for mono- and polyubiquitin. The structure exhibits striking similarities to the Cdc48/p97 N domain. It contains the double-psi beta barrel motif, which is thus identified as a ubiquitin binding domain. Significantly, Ufd1 shows higher affinity toward polyubiquitin than monoubiquitin, attributable to the utilization of separate binding sites with different affinities. Further studies revealed that the Ufd1-ubiquitin interaction involves hydrophobic contacts similar to those in well-characterized ubiquitin binding proteins. Our results provide a structural basis for a previously proposed synergistic binding of polyubiquitin by Cdc48/p97 and Ufd1.     

Dissecting the ER-associated degradation of a misfolded polytopic membrane protein

It remains unclear how misfolded membrane proteins are selected and destroyed during endoplasmic reticulum-associated degradation (ERAD). For example, chaperones are thought to solubilize aggregation-prone motifs, and some data suggest that these proteins are degraded at the ER. To better define how membrane proteins are destroyed, the ERAD of Ste6p(*), a 12 transmembrane protein, was reconstituted. We found that specific Hsp70/40s act before ubiquitination and facilitate Ste6p(*) association with an E3 ubiquitin ligase, suggesting an active role for chaperones. Furthermore, polyubiquitination was a prerequisite for retrotranslocation, which required the Cdc48 complex and ATP. Surprisingly, the substrate was soluble, and extraction was independent of a ubiquitin chain extension enzyme (Ufd2p). However, Ufd2p increased the degree of ubiquitination and facilitated degradation. These data indicate that polytopic membrane proteins can be extracted from the ER, and define the point of action of chaperones and the requirement for Ufd2p during membrane protein quality control.     

A complex of mammalian ufd1 and npl4 links the AAA-ATPase, p97, to ubiquitin and nuclear transport pathways

The AAA-ATPase, p97/Cdc48p, has been implicated in many different pathways ranging from membrane fusion to ubiquitin-dependent protein degradation. Binding of the p47 complex directs p97 to act in the post-mitotic fusion of Golgi membranes. We now describe another binding complex comprising mammalian Ufd1 and Npl4. Yeast Ufd1p is required for ubiquitin-dependent protein degradation whereas yeast Npl4p has been implicated in nuclear transport. In rat liver cytosol, Ufd1 and Npl4 form a binary complex, which exists either alone or bound to p97. Ufd1/Npl4 competes with p47 for binding to p97 and so inhibits Golgi membrane fusion. This suggests that it is involved in another cellular function catalysed by p97, the most likely being ubiquitin-dependent events during mitosis. The fact that the binding of p47 and Ufd1/Npl4 is mutually exclusive suggests that these protein complexes act as adapters, directing a basic p97 activity into different cellular pathways.     

Mechano-chemical effects of Ca(2+) in cross-linked troponin-C films

The ATPase Cdc48 is required for membrane fusion and protein degradation. Recently it has been suggested that Cdc48 in a complex with Ufd1 and Npl4 acts as an ubiquitin-dependent chaperone. Here it is shown that recombinant Cdc48 alone can distinguish between the native and the non-native conformation of model substrates. First, Cdc48 prevents luciferase from aggregating following a heat shock. Second, it inhibits the aggregation of rhodanese upon dilution. Third, Cdc48 binds specifically to heat-denatured luciferase. These chaperone-like functions seem to be independent of ATPase activity. Furthermore, Cdc48 can act as a co-chaperone in the Hsc70–Hsp40 chaperone system. These results show that Cdc48 possesses chaperone-like activities and can functionally interact with Hsc70. Cdc48’s ability to recognise denatured proteins can also be a source of unspecific binding in biochemical interaction experiments.     

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.     

Cdc48p interacts with Ufd3p, a WD repeat protein required for ubiquitin-mediated proteolysis in Saccharomyces cerevisiae.

A library of random 10 residue peptides fused to the N-terminus of a reporter protein was screened in the yeast Saccharomyces cerevisiae for sequences that can target the reporter for degradation by the N-end rule pathway, a ubiquitin (Ub)-dependent proteolytic system that recognizes potential substrates through binding to their destabilizing N-terminal residues. One of the N-terminal sequences identified by this screen was used in a second screen for mutants incapable of degrading the corresponding reporter fusion. A mutant thus identified had an abnormally low content of free Ub. This mutant was found to be allelic to a previously isolated mutant in a Ub-dependent proteolytic system distinct from the N-end rule pathway. We isolated the gene involved, termed UFD3, which encodes an 80 kDa protein containing tandem repeats of a motif that is present in many eukaryotic proteins and called the WD repeat. Both co-immunoprecipitation and two-hybrid assays demonstrated that Ufd3p is an in vivo ligand of Cdc48p, an essential ATPase required for the cell cycle progression and the fusion of endoplasmic reticulum membranes. Further, we showed that, similarly to Ufd3p, Cdc48p is also required for the Ub-dependent proteolysis of test substrates. The discovery of the Ufd3p--Cdc48p complex and the finding that this complex is a part of the Ub system open up a new direction for studies of the function of Ub in the cell cycle and membrane dynamics.     

Cdc48/p97, a key actor in the interplay between autophagy and ubiquitin/proteasome catabolic pathways

The AAA-ATPase Cdc48/p97 controls a large array of cellular functions including protein degradation, cell division, membrane fusion through its ability to interact with and control the fate of ubiquitylated proteins. More recently, Cdc48/p97 also appeared to be involved in autophagy, a catabolic cell response that has long been viewed as completely distinct from the Ubiquitine/Proteasome System. In particular, conjugation by ubiquitin or ubiquitin-like proteins as well as ubiquitin binding proteins such as Cdc48/p97 and its cofactors can target degradation by both catabolic pathways. This review will focus on the recently described functions of Cdc48/p97 in autophagosome biogenesis as well as selective autophagy.     

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.     

Cdc48 and Ufd3, new partners of the ubiquitin protease Ubp3, are required for ribophagy

Ubiquitin‐dependent processes can be antagonized by substrate‐specific deubiquitination enzymes involved in many cellular functions. In this study, we show that the yeast Ubp3–Bre5 deubiquitination complex interacts with both the chaperone‐like Cdc48, a major actor of the ubiquitin and proteasome system, and Ufd3, a ubiquitin‐binding cofactor of Cdc48. We observed that these partners are required for the Ubp3–Bre5‐dependent and starvation‐induced selective degradation of yeast mature ribosomes, also called ribophagy. By contrast, proteasome‐dependent degradation does not participate in this process. Our data favour the idea that these factors cooperate to recognize and deubiquitinate specific substrates of ribophagy before their vacuolar degradation.     

Cdc48-Ufd1-Npl4: stuck in the middle with Ub

The ubiquitin-proteasome pathway has a well-defined beginning and end. Target proteins are initially recognized by upstream components and tagged with polyubiquitin chains. The 26S proteasome then degrades these polyubiquitinated proteins. Until recently, it was not known what, if any, steps occurred between the initial polyubiquitination of target proteins and their final degradation. Several new papers investigating the function of the Cdc48-Ufd1-Npl4 complex indicate that there is indeed a middle to the ubiquitin-proteasome pathway. The Cdc48-Ufd1-Npl4 complex functions in the recognition of several polyubiquitin-tagged proteins and facilitates their presentation to the 26S proteasome for processive degradation or even more specific processing. The elucidation of Cdc48, Ufd1 and Npl4 action not only provides long-sought functions for these specific proteins, but illuminates a poorly understood part of the ubiquitin-proteasome pathway.     

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.