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.     

Cdc48Ufd1/Npl4 segregase removes mislocalized centromeric histone H3 variant CENP-A from non-centromeric chromatin

Restricting the localization of CENP-A (Cse4 in Saccharomyces cerevisiae) to centromeres prevents chromosomal instability (CIN). Mislocalization of overexpressed CENP-A to non-centromeric chromatin contributes to CIN in budding and fission yeasts, flies, and humans. Overexpression and mislocalization of CENP-A is observed in cancers and is associated with increased invasiveness. Mechanisms that remove mislocalized CENP-A and target it for degradation have not been defined. Here, we report that Cdc48 and its cofactors Ufd1 and Npl4 facilitate the removal of mislocalized Cse4 from non-centromeric chromatin. Defects in removal of mislocalized Cse4 contribute to lethality of overexpressed Cse4 in cdc48,ufd1 andnpl4 mutants. High levels of polyubiquitinated Cse4 and mislocalization of Cse4 are observed in cdc48-3, ufd1-2 and npl4-1mutants even under normal physiological conditions, thereby defining polyubiquitinated Cse4 as the substrate of the ubiquitin directed segregase Cdc48^Ufd1/Npl4. Accordingly, Npl4, the ubiquitin binding receptor, associates with mislocalized Cse4, and this interaction is dependent on Psh1-mediated polyubiquitination of Cse4. In summary, we provide the first evidence for a mechanism that facilitates the removal of polyubiquitinated and mislocalized Cse4 from non-centromeric chromatin. Given the conservation of Cdc48^Ufd1/Npl4 in humans, it is likely that defects in such pathways may contribute to CIN in human cancers.     

Concerted Action of the Ubiquitin-Fusion Degradation Protein 1 (Ufd1) and Sumo-Targeted Ubiquitin Ligases (STUbLs) in the DNA-Damage Response

In eukaryotes many players in the DNA-damage response (DDR) catalyze protein sumoylation or ubiquitylation. Emphasis has been placed on how these modifications orchestrate the sequential recruitment of repair factors to sites of DNA damage or stalled replication forks. Here, we shed light on a pathway in which sumoylated factors are eliminated through the coupled action of Sumo-targeted ubiquitin ligases (STUbLs) and the ubiquitin-fusion degradation protein 1 (Ufd1). Ufd1 is a subunit of the Cdc48-Ufd1-Npl4 complex implicated in the sorting of ubiquitylated substrates for degradation by the proteasome. We find that in fission yeast, Ufd1 interacts physically and functionally with the Sumo-targeted ubiquitin ligase (STUbL) Rfp1, homologous to human RNF4, and with the Sumo E3 ligase Pli1, homologous to human PIAS1. Deleting a C-terminal domain of Ufd1 that mediates the interaction of Ufd1 with Rfp1, Pli1, and Sumo (ufd1ΔCt ^213-342) lead to an accumulation of high-molecular-weight Sumo conjugates and caused severe genomic instabilities. The spectrum of sensitivity of ufd1ΔCt ^213-342 cells to genotoxins, the epistatic relationships of ufd1ΔCt ^213-342 with mutations in DNA repair factors, and the localization of the repair factor Rad22 in ufd1ΔCt ^213-342 cells point to ufd1ΔCt ^213-342 cells accumulating aberrant structures during replication that require homologous recombination (HR) for their repair. We present evidence that HR is however often not successful in ufd1ΔCt ^213-342 cells and we identify Rad22 as one of the high-molecular-weight conjugates accumulating in the ufd1ΔCt ^213-342 mutant consistent with Rad22 being a STUbL/Ufd1 substrate. Suggesting a direct role of Ufd1 in the processing of Sumo-conjugates, Ufd1 formed nuclear foci colocalizing with Sumo during the DDR, and Sumo-conjugates accumulated in foci in the ufd1ΔCt ^213-342 mutant. Broader functional relationships between Ufd1 and STUbLs conceivably affect numerous cellular processes beyond the DDR.     

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.     

Structure and Function of the PLAA/Ufd3-p97/Cdc48 Complex

PLAA (ortholog of yeast Doa1/Ufd3, also know as human PLAP or phospholipase A2-activating protein) has been implicated in a variety of disparate biological processes that involve the ubiquitin system. It is linked to the maintenance of ubiquitin levels, but the mechanism by which it accomplishes this is unclear. The C-terminal PUL (PLAP, Ufd3p, and Lub1p) domain of PLAA binds p97, an AAA ATPase, which among other functions helps transfer ubiquitinated proteins to the proteasome for degradation. In yeast, loss of Doa1 is suppressed by altering p97/Cdc48 function indicating that physical interaction between PLAA and p97 is functionally important. Although the overall regions of interaction between these proteins are known, the structural basis has been unavailable. We solved the high resolution crystal structure of the p97-PLAA complex showing that the PUL domain forms a 6-mer Armadillo-containing domain. Its N-terminal extension folds back onto the inner curvature forming a deep ridge that is positively charged with residues that are phylogenetically conserved. The C terminus of p97 binds in this ridge, where the side chain of p97-Tyr⁸⁰⁵, implicated in phosphorylation-dependent regulation, is buried. Expressed in doa1Δ null cells, point mutants of the yeast ortholog Doa1 that disrupt this interaction display slightly reduced ubiquitin levels, but unlike doa1Δ null cells, showed only some of the growth phenotypes. These data suggest that the p97-PLAA interaction is important for a subset of PLAA-dependent biological processes and provides a framework to better understand the role of these complex molecules in the ubiquitin system.     

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.     

The AAA ATPase p97/VCP interacts with its alternative co-factors, Ufd1-Npl4 and p47, through a common bipartite binding mechanism

The AAA ATPase p97/VCP forms complexes with different adapters to fulfill distinct cellular functions. We analyzed the structural organization of the Ufd1-Npl4 adapter complex and its interaction with p97 and compared it with another adapter, p47. We found that the binary Ufd1-Npl4 complex forms a heterodimer that cooperatively interacts with p97 via a bipartite binding mechanism. Binding site 1 (BS1) is a short hydrophobic stretch in the C-terminal domain of Ufd1. The second binding site is located at the N terminus of Npl4 and is activated upon binding of Ufd1 to Npl4. It consists of about 80 amino acids that are predicted to form a ubiquitin fold domain (UBD). Despite the lack of overall homology between Ufd1-Npl4 and p47, both adapters use identical binding mechanisms. Like the ubiquitin fold ubiquitin regulatory X (UBX) domain in p47, the Npl4-UBD interacts with p97 via the loop between its strands 3 and 4 and a conserved arginine in strand 1. Furthermore, we identified a region in p47 homologous to Ufd1-BS1. The UBD/UBX and the BS1 of both adapters interact with p97 independently, whereas homologous binding sites in both adapters compete for binding to p97. In contrast to p47, however, Ufd1-Npl4 does not regulate the ATPase activity of p97; nor does a variant of p47 that contains both binding sites but lacks the N-terminal domains. Therefore, the binding sites alone do not regulate p97 directly but rather serve as anchor points to position adapter-specific domains at critical locations to modulate p97-mediated reactions.     

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.     

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.     

Ubiquitin ligase Ufd2 is required for efficient degradation of Mps1 kinase

Ufd2 is a U-box-containing ubiquitylation enzyme that promotes ubiquitin chain assembly on substrates. The physiological function of Ufd2 remains poorly understood. Here, we show that ubiquitylation and degradation of the cell cycle kinase Mps1, a known target of the anaphase-promoting complex E3, require Ufd2 enzyme. Yeast cells lacking UFD2 exhibit altered chromosome stability and several spindle-related phenotypes, expanding the biological function of Ufd2. We demonstrate that Ufd2-mediated Mps1 degradation is conserved in humans. Our results underscore the significance of Ufd2 in proteolysis and further suggest that Ufd2-like enzymes regulate far more substrates than previously envisioned.     

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.     

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.     

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.     

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.     

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.     

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.     

Analysis of Npl4 deletion mutants in mammalian cells unravels new Ufd1-interacting motifs and suggests a regulatory role of Npl4 in ERAD

Npl4 is a 67 kDa protein forming a stable heterodimer with Ufd1, which in turn binds the ubiquitous p97/VCP ATPase. According to a widely accepted model, VCP^Ufd1–Npl4 promotes the retrotranslocation of emerging ER proteins, their ubiquitination by associated ligases, and handling to the 26S proteasome for degradation in a process known as ERAD (ER-associated degradation). Using a series of Npl4 deletion mutants we have revealed that the binding of Ufd1 to Npl4 is mediated by two regions: a conserved stretch of amino acids from 113 to 255 within the zf-Npl4 domain and by the Npl4 homology domain between amino acids 263 and 344. Within the first region, we have identified two discrete subdomains: one involved in Ufd1 binding and one regulating VCP binding. Expression of any one of the mutants failed to induce any changes in the morphology of the ER or Golgi compartments. Moreover, we have observed that overexpression of all the analyzed mutants induced mild ER stress, as evidenced by increased Grp74/BiP expression without associated XBP1 splicing or induction of apoptosis. Surprisingly, we have not observed any accumulation of the typical ERAD substrate αTCR. This favors the model where the Ufd1–Npl4 dimer forms a regulatory gate at the exit from the retrotranslocone, rather than actively promoting retrotranslocation like the p97VCP ATPase.     

Dual Recruitment of Cdc48 (p97)-Ufd1-Npl4 Ubiquitin-selective Segregase by Small Ubiquitin-like Modifier Protein (SUMO) and Ubiquitin in SUMO-targeted Ubiquitin Ligase-mediated Genome Stability Functions

Protein modification by SUMO and ubiquitin critically impacts genome stability via effectors that "read" their signals using SUMO interaction motifs or ubiquitin binding domains, respectively. A novel mixed SUMO and ubiquitin signal is generated by the SUMO-targeted ubiquitin ligase (STUbL), which ubiquitylates SUMO conjugates. Herein, we determine that the "ubiquitin-selective" segregase Cdc48-Ufd1-Npl4 also binds SUMO via a SUMO interaction motif in Ufd1 and can thus act as a selective receptor for STUbL targets. Indeed, we define key cooperative DNA repair functions for Cdc48-Ufd1-Npl4 and STUbL, thereby revealing a new signaling mechanism involving dual recruitment by SUMO and ubiquitin for Cdc48-Ufd1-Npl4 functions in maintaining genome stability.     

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.     

WD40 repeat propellers define a ubiquitin-binding domain that regulates turnover of F box proteins

WD40-repeat β-propellers are found in a wide range of proteins involved in distinct biological activities. We define a large subset of WD40 β-propellers as a class of ubiquitin-binding domains. Using the β-propeller from Doa1/Ufd3 as a paradigm, we find the conserved top surface of the Doa1 β-propeller binds the hydrophobic patch of ubiquitin centered on residues I44, L8, and V70. Mutations that disrupt ubiquitin binding abrogate Doa1 function, demonstrating the importance of this interaction. We further demonstrate that WD40 β-propellers from a functionally diverse set of proteins bind ubiquitin in a similar fashion. This set includes members of the F box family of SCF ubiquitin E3 ligase adaptors. Using mutants defective in binding, we find that ubiquitin interaction by the F box protein Cdc4 promotes its autoubiquitination and turnover. Collectively, our results reveal a molecular mechanism that may account for how ubiquitin controls a broad spectrum of cellular activities.