Definitive evidence for Ufd2-catalyzed elongation of the ubiquitin chain through Lys48 linkage

Saccharomyces cerevisiae Ufd2 is a ubiquitin chain elongation factor in the ubiquitin fusion degradation (UFD) pathway and functions in stress tolerance. A recent study has suggested that the mammalian Ufd2 homologue UFD2a catalyzes formation of Lys27- and Lys33-linked polyubiquitin chains rather than the Lys48-linked chain, but the linkage type of the polyubiquitin chain formed by yeast Ufd2 remains unclear. To determine the property of Ufd2, we reconstituted the UFD pathway using purified enzymes from yeast. Direct determination of the ubiquitin chain linkage type in polyubiquitinated UFD substrates by MALDI-TOF mass spectrometry revealed that Ufd2 catalyzes elongation of the ubiquitin chain through Lys48 linkage.     

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.     

A synthetic defect in protein degradation caused by loss of Ufd4 and Rad23

The UFD (ubiquitin fusion degradation) pathway is responsible for multiubiquitination of the fusion proteins that bear a "non-removable" N-terminal ubiquitin moiety. Previous reports have shown that the UFD pathway is conserved from yeast to human. The essential elements of the UFD pathway have also been identified in Saccharomyces cerevisiae. These studies, however, are limited to use of engineered UFD substrates. The biological significance of the UFD pathway remains unknown. Here we demonstrate that Ufd4, the E3 component of the UFD pathway, is involved in controlling the degradation of Rad4, a nucleotide excision repair protein. Moreover, simultaneous loss of Ufd4 and Rad23 exhibits a synthetic inhibitory effect on Rad4 degradation, presenting the first example that a UBA/UBL-domain protein functionally overlaps with a ubiquitin ligase in determining the turnover rate of a protein substrate. The current work also provides a direction for further investigation of the physiological functions of the UFD pathway.     

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.     

Identification of components of the murine histone deacetylase 6 complex: link between acetylation and ubiquitination signaling pathways.

The immunopurification of the endogenous cytoplasmic murine histone deacetylase 6 (mHDAC6), a member of the class II HDACs, from mouse testis cytosolic extracts allowed the identification of two associated proteins. Both were mammalian homologues of yeast proteins known to interact with each other and involved in the ubiquitin signaling pathway: p97/VCP/Cdc48p, a homologue of yeast Cdc48p, and phospholipase A2-activating protein, a homologue of yeast UFD3 (ubiquitin fusion degradation protein 3). Moreover, in the C-terminal region of mHDAC6, a conserved zinc finger-containing domain named ZnF-UBP, also present in several ubiquitin-specific proteases, was discovered and was shown to mediate the specific binding of ubiquitin by mHDAC6. By using a ubiquitin pull-down approach, nine major ubiquitin-binding proteins were identified in mouse testis cytosolic extracts, and mHDAC6 was found to be one of them. All of these findings strongly suggest that mHDAC6 could be involved in the control of protein ubiquitination. The investigation of biochemical properties of the mHDAC6 complex in vitro further supported this hypothesis and clearly established a link between protein acetylation and protein ubiquitination.     

Cloning and molecular characterization of three ubiquitin fusion degradation 1 (Ufd1) ortholog genes from Xenopus laevis, Gallus gallus and Drosophila melanogaster.

The yeast ubiquitin fusion degradation 1 (Ufd1) protein is involved in a degradation pathway for ubiquitin fused products. The human ortholog gene (UFD1-like, UFD1L) is deleted in patients affected by the DiGeorge/velocardiofacial syndromes. We report the cloning of UFD1L orthologs from Drosophila melanogaster (dufd1l), Xenopus laevis and Gallus gallus. The 1,125-bp Drosophila cDNA encodes a protein of 316 amino acids, showing 60% identity with the human and murine proteins. The identity to the G. gallus, X. laevis, C. elegans and S. cerevisiae proteins is 95%, 83%, 32%, and 36%, respectively. Northern expression data in Drosophila indicate that dufd1l is expressed through embryonic, larval and pupal development, as well as in the adult fly.     

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.     

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.     

The N-end rule pathway is mediated by a complex of the RING-type Ubr1 and HECT-type Ufd4 ubiquitin ligases

Substrates of the N-end rule pathway are recognized by the Ubr1 E3 ubiquitin ligase through their destabilizing amino-terminal residues. Our previous work showed that the Ubr1 E3 and the Ufd4 E3 together target an internal degradation signal (degron) of the Mgt1 DNA repair protein. Ufd4 is an E3 enzyme of the ubiquitin-fusion degradation (UFD) pathway that recognizes an N-terminal ubiquitin moiety. Here we show that the RING-type Ubr1 E3 and the HECT-type Ufd4 E3 interact, both physically and functionally. Although Ubr1 can recognize and polyubiquitylate an N-end rule substrate in the absence of Ufd4, the Ubr1-Ufd4 complex is more processive in that it produces a longer substrate-linked polyubiquitin chain. Conversely, Ubr1 can function as a polyubiquitylation-enhancing component of the Ubr1-Ufd4 complex in its targeting of UFD substrates. We also found that Ubr1 can recognize the N-terminal ubiquitin moiety. These and related advances unify two proteolytic systems that have been studied separately for two decades.     

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.     

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.     

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.     

Sel1p/Ubx2p participates in a distinct Cdc48p-dependent endoplasmic reticulum-associated degradation pathway

The endoplasmic reticulum (ER) serves a critical role in the biogenesis of secretory proteins. Folding of nascent polypeptides occurs in the ER before anterograde transport through the secretory pathway, whereas terminally misfolded secretory proteins are recognized and eliminated by ER-associated degradation (ERAD). Here, we investigated the role of the ubiquitin regulatory X (UBX) domain-containing protein Sel1p in ER quality control and transport. Mutant sel1Delta yeast displayed a constitutively active unfolded protein response and a mildly reduced rate of secretory protein transport from the ER. Immunoisolation of Sel1p from detergent-solubilized ER microsomes revealed a protein complex containing both Cdc48p and Npl4p and suggested a direct role for Sel1p in ERAD. In cells that lack Sel1p, we observed a reduction in the level of Cdc48p bound to ER membranes and a decrease in the turnover rate of two model ERAD substrates, carboxypeptidase Y* and Ste6*. In addition, we found that Sel1p and a second UBX domain-containing protein, Shp1p, associated with Cdc48p in a mutually exclusive manner. Interestingly, the association of Sel1p with Cdc48p was regulated by ATP, while the interaction of Shp1p with Cdc48p was not influenced by ATP. Based on these findings, we conclude that Sel1p operates in the ERAD pathway by coupling Cdc48p to ER membranes and that Shp1p acts in a distinct Cdc48p-dependent protein degradation pathway.     

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.     

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.     

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.     

Functional regulation of FEZ1 by the U-box-type ubiquitin ligase E4B contributes to neuritogenesis

E4B (also known as UFD2a) is a mammalian homolog of Saccharomyces cerevisiae Ufd2, which was originally described as a ubiquitin chain assembly factor (E4). E4B is a U-box-type ubiquitin-protein isopeptide ligase (E3) and likely functions as either an E3 or an E4. With a yeast two-hybrid screen, we have now identified FEZ1 (fasciculation and elongation protein zeta 1) as a protein that interacts with E4B. FEZ1 is implicated in neuritogenesis when phosphorylated by protein kinase Czeta (PKCzeta). Interaction between E4B and FEZ1 in mammalian cells was enhanced by coexpression of constitutively active PKCzeta. E4B mediated the polyubiquitylation of FEZ1 but did not affect its intracellular stability, suggesting that such modification of FEZ1 is not a signal for its proteolysis. Polyubiquitylation of FEZ1 by E4B required Lys(27) of ubiquitin. Expression of a dominant-negative mutant of E4B in rat pheochromocytoma PC12 cells resulted in inhibition of neurite extension induced either by nerve growth factor or by coexpression of FEZ1 and constitutively active PKCzeta. These findings indicate that E4B serves as a ubiquitin ligase for FEZ1 and thereby regulates its function but not its degradation.