Doa1 targets ubiquitinated substrates for mitochondria-associated degradation

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

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.     

A genomic screen identifies Dsk2p and Rad23p as essential components of ER-associated degradation

We developed a growth test to screen for yeast mutants defective in endoplasmic reticulum (ER) quality control and associated protein degradation (ERAD) using the membrane protein CTL*, a chimeric derivative of the classical ER degradation substrate CPY*. In a genomic screen of approximately 5,000 viable yeast deletion mutants, we identified genes necessary for ER quality control and degradation. Among the new gene products, we identified Dsk2p and Rad23p. We show that these two proteins are probably delivery factors for ubiquitinated ER substrates to the proteasome, following their removal from the membrane via the Cdc48-Ufd1-Npl4p complex. In contrast to the ERAD substrate CTG*, proteasomal degradation of a cytosolic CPY*-GFP fusion is not dependent on Dsk2p and Rad23p, indicating pathway specificity for both proteins. We propose that, in certain degradation pathways, Dsk2p, Rad23p and the trimeric Cdc48 complex function together in the delivery of ubiquitinated proteins to the proteasome, avoiding malfolded protein aggregates in the cytoplasm.     

Use of modular substrates demonstrates mechanistic diversity and reveals differences in chaperone requirement of ERAD

The endoplasmic reticulum (ER) harbors a protein quality control system, which monitors protein folding in the ER. Elimination of malfolded proteins is an important function of this protein quality control. Earlier studies with various soluble and transmembrane ER-associated degradation (ERAD) substrates revealed differences in the ER degradation machinery used. To unravel the nature of these differences we generated two type I membrane ERAD substrates carrying malfolded carboxypeptidase yscY (CPY*) as the ER-luminal ERAD recognition motif. Whereas the first, CT* (CPY*-TM), has no cytoplasmic domain, the second, CTG*, has the green fluorescent protein present in the cytosol. Together with CPY*, these three substrates represent topologically diverse malfolded proteins, degraded via ERAD. Our data show that degradation of all three proteins is dependent on the ubiquitin-proteasome system involving the ubiquitin-protein ligase complex Der3/Hrd1p-Hrd3p, the ubiquitin conjugating enzymes Ubc1p and Ubc7p, as well as the AAA-ATPase complex Cdc48-Ufd1-Npl4 and the 26S proteasome. In contrast to soluble CPY*, degradation of the membrane proteins CT* and CTG* does not require the ER proteins Kar2p (BiP) and Der1p. Instead, CTG* degradation requires cytosolic Hsp70, Hsp40, and Hsp104p chaperones.     

The Cdc48/p97-Ufd1-Npl4 Complex: Its Potential Role in Coordinating Cellular Morphogenesis During the M-G1 Transition

The AAA ATPase Cdc48/p97 together with its adaptors, Ufd1-Npl4, regulate membrane-related functions and mitotic spindle disassembly by directly binding to membrane-associated proteins or spindle assembly factors, modulating their interactions with membranes or spindles, respectively. Here, we discuss the possibility that the Cdc48/p97-Ufd1-Npl4 complex has a more general role in mediating morphological transitions as the cell exits mitosis and enters G1.     

The Cdc48/p97-Ufd1-Npl4 complex: its potential role in coordinating cellular morphogenesis during the M-G1 transition

The AAA ATPase Cdc48/p97 together with its adaptors, Ufd1-Npl4, regulate membrane-related functions and mitotic spindle disassembly by directly binding to membrane-associated proteins or spindle assembly factors, modulating their interactions with membranes or spindles, respectively. Here, we discuss the possibility that the Cdc48/ p97-Ufd1-Npl4 complex has a more general role in mediating morphological transitions as the cell exits mitosis and enters G(1).     

40S subunit dissociation and proteasome-dependent RNA degradation in nonfunctional 25S rRNA decay

Eukaryotic cells have quality control systems that eliminate nonfunctional rRNAs with deleterious mutations (nonfunctional rRNA decay, NRD). We have previously reported that 25S NRD requires an E3 ubiquitin ligase complex, which is involved in ribosomal ubiquitination. However, the degradation process of nonfunctional ribosomes has remained unknown. Here, using genetic screening, we identified two ubiquitin-binding complexes, the Cdc48-Npl4-Ufd1 complex (Cdc48 complex) and the proteasome, as the factors involved in 25S NRD. We show that the nonfunctional 60S subunit is dissociated from the 40S subunit in a Cdc48 complex-dependent manner, before it is attacked by the proteasome. When we examined the nonfunctional 60S subunits that accumulated under proteasome-depleted conditions, the majority of mutant 25S rRNAs retained their full length at a single-nucleotide resolution. This indicates that the proteasome is an essential factor triggering rRNA degradation. We further showed that ribosomal ubiquitination can be stimulated solely by the suppression of the proteasome, suggesting that ubiquitin-proteasome-dependent RNA degradation occurs in broader situations, including in general rRNA turnover.     

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.     

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 Conserved Protein with AN1 Zinc Finger and Ubiquitin-like Domains Modulates Cdc48 (p97) Function in the Ubiquitin-Proteasome Pathway

Regulated protein degradation mediated by the ubiquitin-proteasome system (UPS) is critical to eukaryotic protein homeostasis. Often vital to degradation of protein substrates is their disassembly, unfolding, or extraction from membranes. These processes are catalyzed by the conserved AAA-ATPase Cdc48 (also known as p97). Here we characterize the Cuz1 protein (Cdc48-associated UBL/zinc finger protein-1), encoded by a previously uncharacterized arsenite-inducible gene in budding yeast. Cuz1, like its human ortholog ZFAND1, has both an AN1-type zinc finger (Zf_AN1) and a divergent ubiquitin-like domain (UBL). We show that Cuz1 modulates Cdc48 function in the UPS. The two proteins directly interact, and the Cuz1 UBL, but not Zf_AN1, is necessary for binding to the Cdc48 N-terminal domain. Cuz1 also associates, albeit more weakly, with the proteasome, and the UBL is dispensable for this interaction. Cuz1-proteasome interaction is strongly enhanced by exposure of cells to the environmental toxin arsenite, and in a proteasome mutant, loss of Cuz1 enhances arsenite sensitivity. Whereas loss of Cuz1 alone causes only minor UPS degradation defects, its combination with mutations in the Cdc48^Npl4-Ufd1 complex leads to much greater impairment. Cuz1 helps limit the accumulation of ubiquitin conjugates on both the proteasome and Cdc48, suggesting a possible role in the transfer of ubiquitylated substrates from Cdc48 to the proteasome or in their release from these complexes.     

Function of the p97-Ufd1-Npl4 complex in retrotranslocation from the ER to the cytosol: dual recognition of nonubiquitinated polypeptide segments and polyubiquitin chains

A member of the family of ATPases associated with diverse cellular activities, called p97 in mammals and Cdc48 in yeast, associates with the cofactor Ufd1-Npl4 to move polyubiquitinated polypeptides from the endoplasmic reticulum (ER) membrane into the cytosol for their subsequent degradation by the proteasome. Here, we have studied the mechanism by which the p97-Ufd1-Npl4 complex functions in this retrotranslocation pathway. Substrate binding occurs when the first ATPase domain of p97 (D1 domain) is in its nucleotide-bound state, an interaction that also requires an association of p97 with the membrane through its NH2-terminal domain. The two ATPase domains (D1 and D2) of p97 appear to alternate in ATP hydrolysis, which is essential for the movement of polypeptides from the ER membrane into the cytosol. The ATPase itself can interact with nonmodified polypeptide substrates as they emerge from the ER membrane. Polyubiquitin chains linked by lysine 48 are recognized in a synergistic manner by both p97 and an evolutionarily conserved ubiquitin-binding site at the NH2 terminus of Ufd1. We propose a dual recognition model in which the ATPase complex binds both a nonmodified segment of the substrate and the attached polyubiquitin chain; polyubiquitin binding may activate the ATPase p97 to pull the polypeptide substrate out of the membrane.     

Cdc48 and cofactors Npl4-Ufd1 are important for G1 progression during heat stress by maintaining cell wall integrity in Saccharomyces cerevisiae

The ubiquitin-selective chaperone Cdc48, a member of the AAA (ATPase Associated with various cellular Activities) ATPase superfamily, is involved in many processes, including endoplasmic reticulum-associated degradation (ERAD), ubiquitin- and proteasome-mediated protein degradation, and mitosis. Although Cdc48 was originally isolated as a cell cycle mutant in the budding yeast Saccharomyces cerevisiae, its cell cycle functions have not been well appreciated. We found that temperature-sensitive cdc48-3 mutant is largely arrested at mitosis at 37°C, whereas the mutant is also delayed in G1 progression at 38.5°C. Reporter assays show that the promoter activity of G1 cyclin CLN1, but not CLN2, is reduced in cdc48-3 at 38.5°C. The cofactor npl4-1 and ufd1-2 mutants also exhibit G1 delay and reduced CLN1 promoter activity at 38.5°C, suggesting that Npl4-Ufd1 complex mediates the function of Cdc48 at G1. The G1 delay of cdc48-3 at 38.5°C is a consequence of cell wall defect that over-activates Mpk1, a MAPK family member important for cell wall integrity in response to stress conditions including heat shock. cdc48-3 is hypersensitive to cell wall perturbing agents and is synthetic-sick with mutations in the cell wall integrity signaling pathway. Our results suggest that the cell wall defect in cdc48-3 is exacerbated by heat shock, which sustains Mpk1 activity to block G1 progression. Thus, Cdc48-Npl4-Ufd1 is important for the maintenance of cell wall integrity in order for normal cell growth and division.     

Involvement of the p97-Ufd1-Npl4 complex in the regulated endoplasmic reticulum-associated degradation of inositol 1,4,5-trisphosphate receptors

Inositol 1,4,5-trisphosphate (IP(3)) receptors form tetrameric, IP(3)-gated channels in endoplasmic reticulum membranes that govern the release of Ca(2+) from this organelle. In response to activation of certain G protein-coupled receptors that persistently elevate IP(3) concentration, IP(3) receptors are ubiquitinated and degraded by the ubiquitin-proteasome pathway. IP(3) receptor ubiquitination is mediated by the ubiquitin-conjugating enzyme, (mam)Ubc7, a component of the endoplasmic reticulum-associated degradation pathway. However, the mechanism by which ubiquitinated IP(3) receptors are transferred to the proteasome is not known. Here, we examine this process and show in several mammalian cell types that the ATPase p97 associates with IP(3) receptors in response to hormonal stimuli that induce IP(3) receptor ubiquitination. To examine the functional relevance of the p97 interaction with IP(3) receptors, we stably and specifically reduced p97 protein levels by 62 +/- 3% in Rat-1 fibroblasts using RNA interference. In these cells, endothelin-1-induced IP(3) receptor degradation was markedly retarded and the accumulation of ubiquitinated IP(3) receptors was markedly enhanced. These effects were reversed by expression of exogenous p97. In addition, Ufd1 and Npl4, which complex with p97, also associated with IP(3) receptors upon hormonal stimulation. We conclude that the p97-Ufd1-Npl4 complex couples ubiquitinated IP(3) receptors to proteasomal degradation and, thus, plays a key role in IP(3) receptor processing. These data also establish that the p97-Ufd1-Npl4 complex mediates endoplasmic reticulum-associated degradation in mammalian cells.     

The Cdc48-Ufd1-Npl4 complex is central in ubiquitin-proteasome triggered catabolite degradation of fructose-1,6-bisphosphatase

The switch from gluconeogenesis to glycolysis in yeast has been shown to require ubiquitin-proteasome dependent elimination of the key enzyme fructose-1,6-bisphosphatase (FBPase). Prior to proteasomal degradation, polyubiquitination of the enzyme occurs via the ubiquitin-conjugating enzymes Ubc1, Ubc4, Ubc5 and Ubc8 in conjunction with a novel multi-subunit ubiquitin ligase, the Gid complex. As an additional machinery required for the catabolite degradation process, we identified the trimeric Cdc48^Ufd1-Npl4 complex and the ubiquitin receptors Dsk2 and Rad23. We show that this machinery acts between polyubiquitination of FBPase and its degradation by the proteasome.     

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

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

Cic1, an adaptor protein specifically linking the 26S proteasome to its substrate, the SCF component Cdc4

In eukaryotes, the ubiquitin-proteasome system plays a major role in selective protein breakdown for cellular regulation. Here we report the discovery of a new essential component of this degradation machinery. We found the Saccharomyces cerevisiae protein Cic1 attached to 26S proteasomes playing a crucial role in substrate specificity for proteasomal destruction. Whereas degradation of short-lived test proteins is not affected, cic1 mutants stabilize the F-box proteins Cdc4 and Grr1, substrate recognition subunits of the SCF complex. Cic1 interacts in vitro and in vivo with Cdc4, suggesting a function as a new kind of substrate recruiting factor or adaptor associated with the proteasome.     

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.