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PubMed2005

Delivery of ubiquitinated substrates to protein-unfolding machines.

Recent work has shown that ubiquitination leads to recognition of target proteins by diverse ubiquitin receptors. One family of receptors delivers the ubiquitinated proteins to the proteasome resulting in ATP-dependent substrate unfolding and proteolysis. A related family of ubiquitin-binding proteins seems to recruit ubiquitinated proteins to Cdc48, an ATPase ring complex that can also unfold proteins. Some targets seem to dock at Cdc48 before the proteasome does, in an ordered pathway. The intimate interplay between the proteasome and Cdc48, mediated in part by loosely associated ubiquitin receptors, has important functions in cellular regulation.

Adenosine Triphosphatases↗

PubMed2004

Distinct machinery is required in Saccharomyces cerevisiae for the endoplasmic reticulum-associated degradation of a multispanning membrane protein and a soluble luminal protein.

The folding and assembly of proteins in the endoplasmic reticulum (ER) lumen and membrane are monitored by ER quality control. Misfolded or unassembled proteins are retained in the ER and, if they cannot fold or assemble correctly, ultimately undergo ER-associated degradation (ERAD) mediated by the ubiquitin-proteasome system. Whereas luminal and integral membrane ERAD substrates both require the proteasome for their degradation, the ER quality control machinery for these two classes of proteins likely differs because of their distinct topologies. Here we establish the requirements for the ERAD of Ste6p*, a multispanning membrane protein with a cytosolic mutation, and compare them with those for mutant form of carboxypeptidase Y (CPY*), a soluble luminal protein. We show that turnover of Ste6p* is dependent on the ubiquitin-protein isopeptide ligase Doa10p and is largely independent of the ubiquitin-protein isopeptide ligase Hrd1p/Der3p, whereas the opposite is true for CPY*. Furthermore, the cytosolic Hsp70 chaperone Ssa1p and the Hsp40 co-chaperones Ydj1p and Hlj1p are important in ERAD of Ste6p*, whereas the ER luminal chaperone Kar2p is dispensable, again opposite their roles in CPY* turnover. Finally, degradation of Ste6p*, unlike CPY*, does not appear to require the Sec61p translocon pore but, like CPY*, could depend on the Sec61p homologue Ssh1p. The ERAD pathways for Ste6p* and CPY* converge at a post-ubiquitination, pre-proteasome step, as both require the ATPase Cdc48p. Our results demonstrate that ERAD of Ste6p* employs distinct machinery from that of the soluble luminal substrate CPY* and that Ste6p* is a valuable model substrate to dissect the cellular machinery required for the ERAD of multispanning membrane proteins with a cytosolic mutation.

Adenosine Triphosphatases↗

PubMed2003

The localization and phosphorylation of p47 are important for Golgi disassembly-assembly during the cell cycle.

In mammalian cells, the Golgi apparatus is disassembled at the onset of mitosis and reassembled at the end of mitosis. This disassembly-reassembly is generally believed to be essential for the equal partitioning of Golgi into two daughter cells. For Golgi disassembly, membrane fusion, which is mediated by NSF and p97, needs to be blocked. For the NSF pathway, the tethering of p115-GM130 is disrupted by the mitotic phosphorylation of GM130, resulting in the inhibition of NSF-mediated fusion. In contrast, the p97/p47 pathway does not require p115-GM130 tethering, and its mitotic inhibitory mechanism has been unclear. Now, we have found that p47, which mainly localizes to the nucleus during interphase, is phosphorylated on Serine-140 by Cdc2 at mitosis. The phosphorylated p47 does not bind to Golgi membranes. An in vitro assay shows that this phosphorylation is required for Golgi disassembly. Microinjection of p47(S140A), which is unable to be phosphorylated, allows the cell to keep Golgi stacks during mitosis and has no effect on the equal partitioning of Golgi into two daughter cells, suggesting that Golgi fragmentation-dispersion may not be obligatory for equal partitioning even in mammalian cells.

Adenosine Triphosphatases↗

PubMed2006

RNA interference analysis of Legionella in Drosophila cells: exploitation of early secretory apparatus dynamics.

Legionella pneumophila translocates multiple bacterial effector proteins into host cells to direct formation of a replication vacuole for the bacterium. The emerging consensus is that formation of this compartment involves recruitment of membrane material that traffics between the endoplasmic reticulum (ER) and Golgi. To investigate this model, a targeted approach was used to knock down expression of proteins involved in membrane trafficking, using RNA interference in Drosophila cells. Surprisingly, few single knockdowns of ER-Golgi transport proteins decreased L. pneumophila replication. By analyzing double-stranded RNAs in pairs, combinations were identified that together caused defects in intracellular replication, consistent with the model that membrane traffic funnels into the replication vacuole from multiple sources. In particular, simultaneous depletion of the intermediate compartment and Golgi-tethering factor transport protein particle together with the ER SNARE protein Sec22 reduced replication efficiency, indicating that introduction of lesions at distinct sites in the secretory system reduces replication efficiency. In contrast to knockdowns in secretory traffic, which required multiple simultaneous hits, knockdown of single cytosolic components of ER-associated degradation, including Cdc48/p97 and associated cofactors, was sufficient to inhibit intracellular replication. The requirement for the Cdc48/p97 complex was conserved in mammalian cells, in which replication vacuoles showed intense recruitment of ubiquitinated proteins, the preferred substrates of Cdc48/p97. This complex promoted dislocation of both ubiquitinated proteins and bacterial effectors from the replication vacuole, consistent with the model that maintenance of high-level replication requires surveillance of the vacuole surface. This work demonstrates that L. pneumophila has the ability to gain access to multiple sites in the secretory system and provides the first evidence for a role of the Cdc48/p97 complex in promoting intracellular replication of pathogens and maintenance of replication vacuoles.

Adenosine Triphosphatases↗

PubMed2001

Isolation of cDNA and genomic DNA clones encoding a calmodulin-binding protein related to a family of ATPases involved in cell division and vesicle fusion.

Calmodulin (CaM), a primary Ca2+ receptor in all eukaryotic cells, is a multifunctional protein that functions by interacting with and modulating the activities of a wide variety of target proteins. Identifying and characterizing these CaM-binding target proteins is essential to define the pathways by which Ca(2+)-regulated signals are transduced. An Arabidopsis thaliana L. flower cDNA expression library constructed in lambda ZAPII was screened for CaM-binding proteins with 35S-labeled CaM. A partial cDNA whose encoded protein shares a high level of similarity with yeast CDC48p was isolated. A genomic clone was isolated using the partial length cDNA clone as a probe, and its nucleotide sequence was determined. The genomic DNA sequence was used to design oligonucleotide primers for polymerase-chain-reaction (PCR) experiments that facilitated cloning and reconstructing a full-length, 3.4-kb cDNA clone. The cDNA encodes a 111-kDa CaM-interacting protein (CIP111) containing motifs characteristic of a diverse family of ATPases, including proteins involved in cell cycle regulation, protein degradation, and vesicle-mediated protein transport. A truncated fusion protein encoded by the carboxy-terminal region of CIP111 was produced in Escherichia coli and shown to bind CaM in a Ca(2+)-dependent manner by protein gel blot and affinity chromatography binding assays. Reverse-transcription PCR analyses demonstrated that CIP111 mRNA is expressed in all organs examined including flowers, siliques, floral stalks, leaves, and roots. DNA blot hybridization analyses indicate that a single-copy gene in Arabidopsis is likely to encode CIP111.

Adenosine Triphosphatases↗

PubMed2006

An arginine/lysine-rich motif is crucial for VCP/p97-mediated modulation of ataxin-3 fibrillogenesis.

Arginine/lysine-rich motifs typically function as targeting signals for the translocation of proteins to the nucleus. Here, we demonstrate that such a motif consisting of four basic amino acids in the polyglutamine protein ataxin-3 (Atx-3) serves as a recognition site for the interaction with the molecular chaperone VCP. Through this interaction, VCP modulates the fibrillogenesis of pathogenic forms of Atx-3 in a concentration-dependent manner, with low concentrations of VCP stimulating fibrillogenesis and excess concentrations suppressing it. No such effect was observed with a mutant Atx-3 variant, which does not contain a functional VCP interaction motif. Strikingly, a stretch of four basic amino acids in the ubiquitin chain assembly factor E4B was also discovered to be critical for VCP binding, indicating that arginine/lysine-rich motifs might be generally utilized by VCP for the targeting of proteins. In vivo studies with Drosophila models confirmed that VCP selectively modulates aggregation and neurotoxicity induced by pathogenic Atx-3. Together, these results define the VCP-Atx-3 association as a potential target for therapeutic intervention and suggest that it might influence the progression of spinocerebellar ataxia type 3.

Adenosine Triphosphatases↗

PubMed1995

The higher plant Arabidopsis thaliana encodes a functional CDC48 homologue which is highly expressed in dividing and expanding cells.

We have identified an Arabidopsis thaliana CDC48 gene which, unlike the putative mammalian homologue vasolin-containing protein (VCP), functionally complements Saccharomyces cerevisiae cdc48 mutants. CDC48 is an essential gene in S. cerevisiae and genetic studies suggest a role in spindle pole body separation. Biochemical studies link VCP function to membrane trafficking and signal transduction. We have described the AtCDC48 expression pattern in a multicellular eukaryote; the zones of cell division, expansion and differentiation are physically separated in higher plants, thus allowing the analysis of in situ expression patterns with respect to the state of cell proliferation. AtCDC48 is highly expressed in the proliferating cells of the vegetative shoot, root, floral inflorescence and flowers, and in rapidly growing cells. AtCDC48 mRNA and the encoded protein are up-regulated in the developing microspores and ovules. AtCDC48 expression is down-regulated in most differentiated cell types. AtCDC48p was primarily localized to the nucleus and, during cytokinesis, to the phragmoplast, a site where membrane vesicles are targeted in the deposition of new cell wall materials. This study shows that the essential cell division function of CDC48 has been conserved by, at least, some multicellular eukaryotes and suggests that in higher plants, CDC48 functions in cell division and growth processes.

Adenosine Triphosphatases↗

PubMed2006

Protein domain-domain interactions and requirements for the negative regulation of Arabidopsis CDC48/p97 by the plant ubiquitin regulatory X (UBX) domain-containing protein, PUX1.

CDC48/p97 is an essential AAA-ATPase chaperone that functions in numerous diverse cellular activities through its interaction with specific adapter proteins. The ubiquitin regulatory X (UBX)-containing protein, PUX1, functions to regulate the hexameric structure and ATPase activity of AtCDC48. To characterize the biochemical mechanism of PUX1 action on AtCDC48, we have defined domains of both PUX1 and AtCDC48 that are critical for interaction and oligomer disassembly. Binding of PUX1 to AtCDC48 was mediated through a region containing both the UBX domain and the immediate C-terminal flanking amino acids (UBX-C). Like other UBX domains, the primary binding site for the UBX-C of PUX1 is the N(a) domain of AtCDC48. Alternative plant PUX protein UBX domains also bind AtCDC48 through the N terminus but were found not to be able to substitute for the action imparted by the UBX-C of PUX1 in hexamer disassembly, suggesting unique features for the UBX-C of PUX1. We propose that the PUX1 UBX-C domain modulates a second binding site on AtCDC48 required for the N-terminal domain of PUX1 to interact with and promote dissociation of the AtCDC48 hexamer. Utilizing Atcdc48 ATP hydrolysis and binding mutants, we demonstrate that PUX1 binding was not affected but that hexamer disassembly was significantly influenced by the ATP status of AtCDC48. ATPase activity in both the D1 and the D2 domains was critical for PUX1-mediated AtCDC48 hexamer disassembly. Together these results provide new mechanistic insight into how the hexameric status and ATPase activity of AtCDC48 are modulated.

Adenosine Triphosphatases↗

PubMed2006

MMI1 (YKL056c, TMA19), the yeast orthologue of the translationally controlled tumor protein (TCTP) has apoptotic functions and interacts with both microtubules and mitochondria.

The yeast orthologue of mammalian TCTP is here proposed to be named Mmi1p (microtubule and mitochondria interacting protein). This protein displays about 50% amino acid sequence identity with its most distantly related orthologs in higher organisms and therefore probably belongs to a small class of yeast proteins which have housekeeping but so far incompletely known functions needed for every eukaryotic cell. Previous investigations of the protein in both higher cells and yeast revealed that it is highly expressed during active growth, but transcriptionally down-regulated in several kinds of stress situations including starvation stress. In human cells, TCTP presumably has anti-apoptotic functions as it binds to Bcl-XL in vivo. TCTP of higher cells was also shown to interact with the translational machinery. It has acquired an additional function in the mammalian immune system, as it is identical with the histamine releasing factor. Here, we show that in S. cerevisiae induction of apoptosis by mild oxidative stress, replicative ageing or mutation of cdc48 leads to translocation of Mmi1p from the cytoplasm to the mitochondria. Mmi1p is stably but reversibly attached to the outer surface of the mitochondria and can be removed by digestion with proteinase K. Glutathionylation of Mmi1p, which is also induced by oxidants, is not a prerequisite or signal for translocation as shown by replacing the only cysteine of Mmi1p by serine. Mmi1p probably interacts with yeast microtubules as deletion of the gene confers sensitivity to benomyl. Conversely, the deletion mutant displays resistance to hydrogen peroxide stress and shows a small but significant elongation of the mother cell-specific lifespan. Our results so far indicate that Mmi1p is one of the few proteins establishing a functional link between microtubules and mitochondria which may be needed for correct localization of mitochondria during cell division.

Adenosine Triphosphatases↗

PubMed2001

Ubiquitin enters the new millennium.

The latest advances in ubiquitin-mediated signaling were discussed at a recent FASEB meeting in Vermont. New findings show that besides signaling proteolysis, ubiquitination can be a signal for trafficking, kinase activation, and other nonproteolytic fates.

Adenosine Triphosphatases↗

PubMed2001

Regulation of organelle membrane fusion by Pkc1p.

Membrane fusion relies on complex protein machineries, which act in sequence to catalyze the fusion of bilayers. The fusion of endoplasmic reticulum membranes requires the t-SNARE Ufe1p, and the AAA ATPase p97/Cdc48p. While the mechanisms of membrane fusion events have begun to emerge, little is known about how this fusion process is regulated. We provide first evidence that endoplasmic reticulum membrane fusion in yeast is regulated by the action of protein kinase C. Specifically, Pkc1p kinase activity is needed to protect the fusion machinery from ubiquitin-mediated degradation.

Adenosine Triphosphatases↗

PubMed2004

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.

Adenosine Triphosphatases↗

PubMed2005

Glycoprotein-specific ubiquitin ligases recognize N-glycans in unfolded substrates.

Misfolded or unassembled polypeptides in the endoplasmic reticulum (ER) are retro-translocated into the cytosol and degraded by the ubiquitin-proteasome system. We reported previously that the SCF(Fbs1,2) ubiquitin-ligase complexes that contribute to ubiquitination of glycoproteins are involved in the ER-associated degradation pathway. Here we investigated how the SCF(Fbs1,2) complexes interact with unfolded glycoproteins. The SCF(Fbs1) complex was associated with p97/VCP AAA ATPase and bound to integrin-beta1, one of the SCF(Fbs1) substrates, in the cytosol in a manner dependent on p97 ATPase activity. Both Fbs1 and Fbs2 proteins interacted with denatured glycoproteins, which were modified with not only high-mannose but also complex-type oligosaccharides, more efficiently than native proteins. Given that Fbs proteins interact with innermost chitobiose in N-glycans, we propose that Fbs proteins distinguish native from unfolded glycoproteins by sensing the exposed chitobiose structure.

Adenosine Triphosphatases↗

PubMed1999

The Janus face of the archaeal Cdc48/p97 homologue VAT: protein folding versus unfolding.

Members of the AAA family of ATPases have been implicated in chaperone-like activities. We used the archaeal Cdc48/p97 homologue VAT as a model system to investigate the effect of an AAA protein on the folding and unfolding of two well-studied, heterologous substrates, cyclophilin and penicillinase. We found that, depending on the Mg2+ concentration, VAT assumes two states with maximum rates of ATP hydrolysis that differ by an order of magnitude. In the low-activity state, VAT accelerated the refolding of penicillinase, whereas in the high-activity state, it accelerated its unfolding. Both reactions were ATP-dependent. In its interaction with cyclophilin, VAT was ATP-independent and only promoted refolding. The N-terminal domain of VAT, which lacks ATPase activity, also accelerated the refolding of cyclophilin but showed no effect on penicillinase. VAT appears to be structurally equivalent over its entire length to Sec18/NSF, suggesting that these results apply more broadly to group II AAA proteins.

Adenosine Triphosphatases↗

PubMed2026

Induced degradation of Ufd1 reveals regulation of cohesin by the VCP/p97Ufd1-Npl4 complex.

The AAA ATPase VCP/p97 has emerged as a critical regulator of ubiquitin and chromatin-associated processes but progress in understanding has been hampered by the complexity of p97 functions and the various p97 cofactors involved. Here, we combined ubiquitin profiling with acutely induced degradation of the Ufd1 subunit of the p97 ubiquitin adapter, Ufd1-Npl4, in human cells. We identified a set of chromatin regulators, HUS1, XRCC1, MORF4L1, and the cohesin subunit RAD21 as targets of p97Ufd1-Npl4 We find that RAD21 is ubiquitylated and targeted by p97Ufd1-Npl4 specifically in S phase to remove a subpopulation of cohesin from chromatin. Acute degradation of Ufd1 in S phase, after replication licensing is completed, impedes replication and leads to replication-associated DNA damage. Our findings suggest that a fraction of cohesin rings need to be removed by p97Ufd1-Npl4 from DNA to allow unhindered replication and reveal a critical function of p97 that ensures genome stability.

Cell Cycle Proteins↗

PubMed1995

Membrane fusion and the cell cycle: Cdc48p participates in the fusion of ER membranes.

The fusion of endoplasmic reticulum (ER) membranes in yeast is an essential process required for normal progression of the nuclear cell cycle, karyogamy, and the maintenance of an intact organellar compartment. We showed previously that this process requires a novel fusion machinery distinct from the classic membrane docking/fusion machinery containing Sec17p (alpha-SNAP) and Sec18p (NSF). Here we show that Cdc48p, a cell-cycle protein with homology to Sec18p, is required in ER fusion. A temperature-sensitive cdc48 mutant is conditionally defective in ER fusion in vitro. Addition of purified Cdc48p restores the fusion of isolated cdc48 mutant ER membranes. We propose that Cdc48p is part of an evolutionarily conserved fusion/docking machinery involved in multiple homotypic fusion events.

Adenosine Triphosphatases↗

PubMed1994

Similarity between putative ATP-binding sites in land plant plastid ORF2280 proteins and the FtsH/CDC48 family of ATPases.

Plastid ORF2280 proteins from five species of land plant are shown to have limited amino-acid sequence similarity to a family of proteins that includes the yeast CDC48, SEC18, PAS1 and SUG1 proteins, three subunits of the mammalian 26S protease, and the Escherichia coli FtsH protein. These proteins all contain one or two ATPase domains and many are involved in cell division, transport of proteins across membranes, or proteolysis. Similarity with the ORF2280 proteins is restricted to a single region of about 130 amino acids that contains: (1) sequences resembling a nucleotide binding site but lacking two normally conserved residues, and (2) a downstream conserved motif with the consensus sequence VIX2TX2PX3DPALX2P. Most of the rest of ORF2280 is very poorly conserved among land plants, even though other family members such as CDC48 have slow rates of protein sequence evolution. In contrast, a protein encoded by plastid DNA of the rhodophyte alga Porphyra purpurea is very similar to E. coli FtsH. Phylogenetic analysis suggests that the red and green plastid genes are not true homologues (orthologues) but distinct members of an ancient gene family.

ATP-Dependent Proteases↗

PubMed2005

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.

Adenosine Triphosphatases↗