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1.
Proc Natl Acad Sci U S A ; 109(4): 1098-103, 2012 Jan 24.
Article in English | MEDLINE | ID: mdl-22232657

ABSTRACT

p97 is a key regulator of numerous cellular pathways and associates with ubiquitin-binding adaptors to remodel ubiquitin-modified substrate proteins. How adaptor binding to p97 is coordinated and how adaptors contribute to substrate remodeling is unclear. Here we present the 3D electron cryomicroscopy reconstructions of the major Ufd1-Npl4 adaptor in complex with p97. Our reconstructions show that p97-Ufd1-Npl4 is highly dynamic and that Ufd1-Npl4 assumes distinct positions relative to the p97 ring upon addition of nucleotide. Our results suggest a model for substrate remodeling by p97 and also explains how p97-Ufd1-Npl4 could form other complexes in a hierarchical model of p97-cofactor assembly.


Subject(s)
Adenosine Triphosphatases/ultrastructure , Carrier Proteins/ultrastructure , Cell Cycle Proteins/ultrastructure , Models, Molecular , Multiprotein Complexes/ultrastructure , Protein Conformation , Proteins/ultrastructure , Cryoelectron Microscopy/methods , Escherichia coli , Image Processing, Computer-Assisted , Imaging, Three-Dimensional , Intercellular Signaling Peptides and Proteins , Intracellular Signaling Peptides and Proteins , Valosin Containing Protein
2.
J Cell Biol ; 191(3): 615-29, 2010 Nov 01.
Article in English | MEDLINE | ID: mdl-21041450

ABSTRACT

Caveolae are long-lived plasma membrane microdomains composed of caveolins, cavins, and a cholesterol-rich membrane. Little is known about how caveolae disassemble and how their coat components are degraded. We studied the degradation of caveolin-1 (CAV1), a major caveolar protein, in CV1 cells. CAV1 was degraded very slowly, but turnover could be accelerated by compromising caveolae assembly. Now, CAV1 became detectable in late endosomes (LE) and lysosomes where it was degraded. Targeting to the degradative pathway required ubiquitination and the endosomal sorting complex required for transport (ESCRT) machinery for inclusion into intralumenal vesicles in endosomes. A dual-tag strategy allowed us to monitor exposure of CAV1 to the acidic lumen of individual, maturing LE in living cells. Importantly, we found that "caveosomes," previously described by our group as independent organelles distinct from endosomes, actually correspond to late endosomal compartments modified by the accumulation of overexpressed CAV1 awaiting degradation. The findings led us to a revised model for endocytic trafficking of CAV1.


Subject(s)
Caveolin 1/metabolism , Lysosomes/metabolism , Ubiquitinated Proteins/metabolism , Cell Line , Endosomal Sorting Complexes Required for Transport/metabolism , HeLa Cells , Humans , Ubiquitination
3.
Nature ; 450(7173): 1258-62, 2007 Dec 20.
Article in English | MEDLINE | ID: mdl-18097415

ABSTRACT

During division of metazoan cells, the nucleus disassembles to allow chromosome segregation, and then reforms in each daughter cell. Reformation of the nucleus involves chromatin decondensation and assembly of the double-membrane nuclear envelope around the chromatin; however, regulation of the process is still poorly understood. In vitro, nucleus formation requires p97 (ref. 3), a hexameric ATPase implicated in membrane fusion and ubiquitin-dependent processes. However, the role and relevance of p97 in nucleus formation have remained controversial. Here we show that p97 stimulates nucleus reformation by inactivating the chromatin-associated kinase Aurora B. During mitosis, Aurora B inhibits nucleus reformation by preventing chromosome decondensation and formation of the nuclear envelope membrane. During exit from mitosis, p97 binds to Aurora B after its ubiquitylation and extracts it from chromatin. This leads to inactivation of Aurora B on chromatin, thus allowing chromatin decondensation and nuclear envelope formation. These data reveal an essential pathway that regulates reformation of the nucleus after mitosis and defines ubiquitin-dependent protein extraction as a common mechanism of Cdc48/p97 activity also during nucleus formation.


Subject(s)
Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/metabolism , Cell Nucleus/metabolism , Chromatin/enzymology , Protein Serine-Threonine Kinases/metabolism , Adenosine Triphosphatases/deficiency , Adenosine Triphosphatases/genetics , Animals , Aurora Kinases , Caenorhabditis elegans , Cell Cycle Proteins/genetics , Cell Nucleus/enzymology , Female , Male , Nuclear Envelope/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , RNA Interference , Ubiquitin/metabolism , Ubiquitination , Valosin Containing Protein , Xenopus laevis
4.
J Cell Sci ; 120(Pt 16): 2895-903, 2007 Aug 15.
Article in English | MEDLINE | ID: mdl-17666429

ABSTRACT

Despite the progress in understanding nuclear envelope (NE) reformation after mitosis, it has remained unclear what drives the required membrane fusion and how exactly this is coordinated with nuclear pore complex (NPC) assembly. Here, we show that, like other intracellular fusion reactions, NE fusion in Xenopus laevis egg extracts is mediated by SNARE proteins that require activation by NSF. Antibodies against Xenopus NSF, depletion of NSF or the dominant-negative NSF(E329Q) variant specifically inhibited NE formation. Staging experiments further revealed that NSF was required until sealing of the envelope was completed. Moreover, excess exogenous alpha-SNAP that blocks SNARE function prevented membrane fusion and caused accumulation of non-flattened vesicles on the chromatin surface. Under these conditions, the nucleoporins Nup107 and gp210 were fully recruited, whereas assembly of FxFG-repeat-containing nucleoporins was blocked. Together, we define NSF- and SNARE-mediated membrane fusion events as essential steps during NE formation downstream of Nup107 recruitment, and upstream of membrane flattening and completion of NPC assembly.


Subject(s)
Membrane Fusion , Nuclear Envelope/metabolism , Nuclear Pore/metabolism , Ovum/metabolism , SNARE Proteins/metabolism , Xenopus Proteins/metabolism , Xenopus laevis/metabolism , Animals , Cell Extracts , Membrane Proteins/metabolism , Mutant Proteins/metabolism , Nuclear Envelope/ultrastructure , Nuclear Proteins/metabolism , Ovum/cytology , Ovum/ultrastructure , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/ultrastructure , ran GTP-Binding Protein/metabolism
5.
J Biol Chem ; 282(29): 21361-9, 2007 Jul 20.
Article in English | MEDLINE | ID: mdl-17491009

ABSTRACT

The AAA ATPase, p97, achieves its versatility through binding to a wide range of cofactor proteins that adapt it to different cellular functions. The heterodimer UN (comprising Ufd1 and Npl4) is an adaptor complex that recruits p97 for numerous tasks, many of which involve the ubiquitin pathway. Insights into the structural specificity of p97 for its UN adaptor are currently negligible. Here, we present the solution structure of the Npl4 "ubiquitin-like" domain (UBD), which adopts a beta-grasp fold with a 3(10) helical insert. Moreover we performed a chemical shift perturbation analysis of its binding surface with the p97 N domain. We assigned the backbone amides of the p97 N domain and probed both its reciprocal binding surface with Npl4 UBD and its interaction with the p97-binding region of Ufd1. NMR data recorded on a 400-kDa full-length UN-hexamer p97 complex reveals an identical mode of interaction. We calculated a structural model for the p97 N-Npl4 UBD complex, and a comparison with the p97-p47 adaptor complex reveals subtle differences in p97 adaptor recognition and specificity.


Subject(s)
Adenosine Triphosphatases/physiology , Nuclear Proteins/physiology , Proteins/physiology , Adenosine Triphosphatases/chemistry , Amino Acid Sequence , Animals , Arabidopsis/metabolism , Escherichia coli/metabolism , Intercellular Signaling Peptides and Proteins , Intracellular Signaling Peptides and Proteins , Mice , Models, Molecular , Molecular Sequence Data , Nuclear Proteins/chemistry , Protein Binding , Protein Conformation , Protein Folding , Protein Structure, Secondary , Protein Structure, Tertiary
6.
Proc Natl Acad Sci U S A ; 104(2): 467-72, 2007 Jan 09.
Article in English | MEDLINE | ID: mdl-17202270

ABSTRACT

p97/VCP (Cdc48 in yeast) is an essential and abundant member of the AAA+ family of ATPases and is involved in a number of diverse cellular pathways through interactions with different adaptor proteins. The two most characterized adaptors for p97 are p47 and the Ufd1 (ubiquitin fusion degradation 1)-Npl4 (nuclear protein localization 4) complex. p47 directs p97 to membrane fusion events and has been shown to be involved in protein degradation. The Ufd1-Npl4 complex directs p97 to an essential role in endoplasmic reticulum-associated degradation and an important role in mitotic spindle disassembly postmitosis. Here we describe the structural features of the Ufd1-Npl4 complex and its interaction with p97 with the aid of EM and other biophysical techniques. The Ufd1-Npl4 heterodimer has an elongated bilobed structure that is approximately 80 x 30 A in dimension. One Ufd1-Npl4 heterodimer is shown to interact with one p97 hexamer to form the p97-Ufd1-Npl4 complex. The Ufd1-Npl4 heterodimer emanates from one region on the periphery of the N-D1 plane of the p97 hexamer. Intriguingly, the p97-p47 and the p97-Ufd1-Npl4 complexes are significantly different in stoichiometry, symmetry, and quaternary arrangement, reflecting their specific actions and their ability to interact with additional cofactors that cooperate with p97 in diverse cellular pathways.


Subject(s)
Adenosine Triphosphatases/chemistry , Nuclear Pore Complex Proteins/chemistry , Nuclear Proteins/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/ultrastructure , Dimerization , Endoplasmic Reticulum/chemistry , Microscopy, Electron , Models, Molecular , Multiprotein Complexes , Nuclear Pore Complex Proteins/genetics , Nuclear Pore Complex Proteins/metabolism , Nuclear Pore Complex Proteins/ultrastructure , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nuclear Proteins/ultrastructure , Nucleocytoplasmic Transport Proteins , Protein Binding , Protein Structure, Quaternary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/ultrastructure , Ubiquitin/metabolism , Vesicular Transport Proteins
7.
Biochim Biophys Acta ; 1744(3): 481-92, 2005 Jul 10.
Article in English | MEDLINE | ID: mdl-16038055

ABSTRACT

The Golgi apparatus in animal cells breaks down at the onset of mitosis and is later rebuilt in the two daughter cells. Two AAA ATPases, NSF and p97/VCP, have been implicated in regulating membrane fusion steps that lead to regrowth of Golgi cisternae from mitotic fragments. NSF dissociates complexes of SNARE proteins, thereby reactivating them to mediate membrane fusion. However, NSF has a second function in regulating SNARE pairing together with the ubiquitin-like protein GATE-16. p97/VCP, on the other hand, is involved in a cycle of ubiquitination and deubiquitination of an unknown target that governs Golgi membrane dynamics. Here, these findings are reviewed and discussed in the context of the increasingly evident role of ubiquitin in membrane traffic processes.


Subject(s)
Adenosine Triphosphatases/metabolism , Golgi Apparatus/metabolism , Mitosis/physiology , Ubiquitin/metabolism , Animals , Catalysis , Cell Cycle Proteins/metabolism , Humans , Membrane Fusion , Models, Biological , SNARE Proteins , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins , Valosin Containing Protein , Vesicular Transport Proteins/metabolism
8.
Biochim Biophys Acta ; 1744(2): 108-19, 2005 Jun 30.
Article in English | MEDLINE | ID: mdl-15878210

ABSTRACT

The Golgi apparatus in animal cells breaks down at the onset of mitosis and is later rebuilt in the two daughter cells. Two AAA ATPases, NSF and p97/VCP, have been implicated in regulating membrane fusion steps that lead to regrowth of Golgi cisternae from mitotic fragments. NSF dissociates complexes of SNARE proteins, thereby reactivating them to mediate membrane fusion. However, NSF has a second function in regulating SNARE pairing together with the ubiquitin-like protein GATE-16. p97/VCP, on the other hand, is involved in a cycle of ubiquitination and deubiquitination of an unknown target that governs Golgi membrane dynamics. Here, these findings are reviewed and discussed in the context of the increasingly evident role of ubiquitin in membrane traffic processes.


Subject(s)
Adenosine Triphosphatases/metabolism , Golgi Apparatus/metabolism , Mitosis/physiology , Ubiquitin/metabolism , Animals , Catalysis , Cell Cycle Proteins/metabolism , Humans , Membrane Fusion , Models, Biological , SNARE Proteins , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins , Valosin Containing Protein , Vesicular Transport Proteins/metabolism
9.
J Biol Chem ; 279(48): 49609-16, 2004 Nov 26.
Article in English | MEDLINE | ID: mdl-15371428

ABSTRACT

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.


Subject(s)
Carrier Proteins/metabolism , Cell Cycle Proteins/metabolism , Proteins/metabolism , Vesicular Transport Proteins/metabolism , Adenosine Triphosphatases , Animals , Binding Sites , Down-Regulation , Mutation , Nuclear Pore Complex Proteins/genetics , Nuclear Pore Complex Proteins/metabolism , Nucleocytoplasmic Transport Proteins , Protein Binding , Protein Structure, Tertiary , Rats , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins , Valosin Containing Protein
10.
J Cell Biol ; 164(7): 973-8, 2004 Mar 29.
Article in English | MEDLINE | ID: mdl-15037600

ABSTRACT

The AAA-ATPase p97/Cdc48 functions in different cellular pathways using distinct sets of adapters and other cofactors. Together with its adaptor Ufd1-Npl4, it extracts ubiquitylated substrates from the membrane for subsequent delivery to the proteasome during ER-associated degradation. Together with its adaptor p47, on the other hand, it regulates several membrane fusion events, including reassembly of Golgi cisternae after mitosis. The finding of a ubiquitin-binding domain in p47 raises the question as to whether the ubiquitin-proteasome system is also involved in membrane fusion events. Here, we show that p97-p47-mediated reassembly of Golgi cisternae requires ubiquitin, but is not dependent on proteasome-mediated proteolysis. Instead, it requires the deubiquitinating activity of one of its cofactors, VCIP135, which reverses a ubiquitylation event that occurs during mitotic disassembly. Together, these data reveal a cycle of ubiquitylation and deubiquitination that regulates Golgi membrane dynamics during mitosis. Furthermore, they represent the first evidence for a proteasome-independent function of p97/Cdc48.


Subject(s)
Carrier Proteins/metabolism , Endopeptidases , Golgi Apparatus/ultrastructure , Mitosis/physiology , Ubiquitin/metabolism , Adenosine Triphosphatases/metabolism , Animals , Carrier Proteins/genetics , Cloning, Molecular , Cysteine Endopeptidases/metabolism , Cytosol/enzymology , Liver/physiology , Membrane Fusion , Molecular Sequence Data , Multienzyme Complexes/metabolism , Nuclear Proteins/metabolism , Proteasome Endopeptidase Complex , Rats , Recombinant Proteins/metabolism
11.
EMBO J ; 23(7): 1411-21, 2004 Apr 07.
Article in English | MEDLINE | ID: mdl-15029239

ABSTRACT

Ubiquitin (Ub) functions in many different biological pathways, where it typically interacts with proteins that contain modular Ub recognition domains. One such recognition domain is the Npl4 zinc finger (NZF), a compact zinc-binding module found in many proteins that function in Ub-dependent processes. We now report the solution structure of the NZF domain from Npl4 in complex with Ub. The structure reveals that three key NZF residues (13TF14/M25) surrounding the zinc coordination site bind the hydrophobic 'Ile44' surface of Ub. Mutations in the 13TF14/M25 motif inhibit Ub binding, and naturally occurring NZF domains that lack the motif do not bind Ub. However, substitution of the 13TF14/M25 motif into the nonbinding NZF domain from RanBP2 creates Ub-binding activity, demonstrating the versatility of the NZF scaffold. Finally, NZF mutations that inhibit Ub binding by the NZF domain of Vps36/ESCRT-II also inhibit sorting of ubiquitylated proteins into the yeast vacuole. Thus, the NZF is a versatile protein recognition domain that is used to bind ubiquitylated proteins during vacuolar protein sorting, and probably many other biological processes.


Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/metabolism , Protein Structure, Secondary , Ubiquitin/metabolism , Zinc Fingers , Amino Acid Sequence , Animals , Carrier Proteins/genetics , Humans , Models, Molecular , Molecular Sequence Data , Multiprotein Complexes , Mutation , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Rats , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Ubiquitin/chemistry , Ubiquitin/genetics
12.
Cell ; 115(3): 355-67, 2003 Oct 31.
Article in English | MEDLINE | ID: mdl-14636562

ABSTRACT

Spindle disassembly at the end of mitosis is a complex and poorly understood process. Here, we report that the AAA-ATPase Cdc48/p97 and its adapters Ufd1-Npl4, which have a well-established role in membrane functions, also regulate spindle disassembly by modulating microtubule dynamics and bundling at the end of mitosis. In the absence of p97-Ufd1-Npl4 function, microtubules in Xenopus egg extracts remain as monopolar spindles attached to condensed chromosomes after Cdc2 kinase activity has returned to the interphase level. Consequently, interphase microtubule arrays and nuclei are not established. Genetic analyses of Cdc48, the yeast homolog of p97, reveal that Cdc48 is also required for disassembly of mitotic spindles after execution of the mitotic exit pathway. Furthermore, Cdc48/p97-Ufd1-Npl4 directly binds to spindle assembly factors and regulates their interaction with microtubules at the end of mitosis. Therefore, Cdc48/p97-Ufd1-Npl4 is an essential chaperone that regulates transformation of the microtubule structure as cells reenter interphase.


Subject(s)
Adenosine Triphosphatases/metabolism , Mitosis , Nuclear Proteins/metabolism , Spindle Apparatus/enzymology , Spindle Apparatus/metabolism , Xenopus Proteins/metabolism , Animals , Cell Cycle Proteins/metabolism , Cyclin B/metabolism , Interphase , Macromolecular Substances , Microtubule-Associated Proteins/metabolism , Oocytes/cytology , Oocytes/enzymology , Protein Binding , RNA-Binding Proteins , Saccharomyces cerevisiae Proteins/metabolism , Valosin Containing Protein , Xenopus laevis
13.
J Cell Biol ; 162(1): 71-84, 2003 Jul 07.
Article in English | MEDLINE | ID: mdl-12847084

ABSTRACT

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.


Subject(s)
Adenosine Triphosphatases/metabolism , Cytosol/enzymology , Endoplasmic Reticulum/enzymology , Eukaryotic Cells/enzymology , Nuclear Pore Complex Proteins/metabolism , Nuclear Proteins/metabolism , Protein Transport/genetics , Saccharomyces cerevisiae Proteins/metabolism , Yeasts/enzymology , Adenosine Triphosphatases/genetics , Adenosine Triphosphate/metabolism , Cells, Cultured , Cysteine Endopeptidases/genetics , Cytosol/ultrastructure , Endoplasmic Reticulum/ultrastructure , Eukaryotic Cells/cytology , Intracellular Membranes/metabolism , Lysine/metabolism , Models, Molecular , Multienzyme Complexes/genetics , Mutation/genetics , Nuclear Pore Complex Proteins/genetics , Nuclear Proteins/genetics , Nucleocytoplasmic Transport Proteins , Peptides/metabolism , Proteasome Endopeptidase Complex , Protein Binding/physiology , Protein Structure, Tertiary/physiology , Saccharomyces cerevisiae Proteins/genetics , Ubiquitin/metabolism , Vesicular Transport Proteins , Yeasts/cytology
14.
J Biol Chem ; 278(22): 20225-34, 2003 May 30.
Article in English | MEDLINE | ID: mdl-12644454

ABSTRACT

Ubiquitylated proteins are directed into a large number of different cellular pathways through interactions with effector proteins that contain conserved ubiquitin binding motifs. Here, we report the solution structure and ubiquitin binding properties of one such motif, the Npl4 zinc finger or RanBP2/Nup358 zinc finger (NZF) domain. Npl4 NZF forms a compact module composed of four antiparallel beta-strands linked by three ordered loops. A single zinc ion is coordinated by four conserved cysteines from the first and third loops, which form two rubredoxin knuckles. Npl4 NZF binds specifically, but weakly, to free ubiquitin using a conserved 13TF14 dipeptide to interact with the "Ile-44" surface of ubiquitin. Our studies reveal the structure of this versatile class of protein binding domains and provide a means for identifying the subset of NZF domains likely to bind ubiquitin.


Subject(s)
Proteins/metabolism , Ubiquitin/metabolism , Zinc Fingers , Animals , Electron Probe Microanalysis , Models, Molecular , Mutagenesis, Site-Directed , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Conformation , Proteins/chemistry , Rats , Zinc/metabolism
15.
EMBO J ; 21(21): 5645-52, 2002 Nov 01.
Article in English | MEDLINE | ID: mdl-12411482

ABSTRACT

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.


Subject(s)
Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport/metabolism , Cell Cycle Proteins/metabolism , Ubiquitin/metabolism , Adaptor Proteins, Vesicular Transport/chemistry , Adenosine Triphosphatases , Amino Acid Motifs , Amino Acid Sequence , Animals , Binding Sites , Golgi Apparatus/metabolism , Mammals , Molecular Sequence Data , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Shc Signaling Adaptor Proteins , Valosin Containing Protein , Zinc Fingers
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