ATPase Activity of p97-Valosin-containing Protein (VCP)

The 97-kDa valosin-containing protein (p97-VCP) plays a role in a wide variety of cellular activities, many of which are regulated by the ubiquitin-proteasome (Ub-Pr)-mediated degradation pathway. We previously demonstrated that VCP binds to multi-ubiquitin chains and may act as a molecular chaperone that targets the ubiquitinated substrates to the proteasome for degradation. In this report, we show that although the ubiquitin chain-binding activity, carried out by the N-terminal 200 residues (N domain), is necessary for the degradation of proteasome substrates, it is not sufficient. Using in vitro degradation assays, we demonstrated that the entire VCP molecule, consisting of the N domain and two ATPase domains D1 and D2, is required for mediating the Ub-Pr degradation. The ATPase activity of VCP requires Mg2+, and is stimulated by high temperature. Under optimal conditions, VCP hydrolyzes ATP with a K m of ∼0.33 mmand a V max of ∼0.52 nmol Pi min−1 μg−1. At a physiological temperature, mutation in D2 significantly inhibits the ATPase activity, while that in D1 has little effect. Interestingly, mutations in D1, but not D2, abolish the heat-stimulated ATPase activity. Thus, we provide the first demonstration that the ATPase activity of VCP is required for mediating the Ub-Pr degradation, that D2 accounts for the major ATPase activity, and that D1 contributes to the heat-induced activity.

[1]  S. Thoms.  Cdc48 can distinguish between native and non‐native proteins in the absence of cofactors , 2002, FEBS letters.

[2]  S. Jentsch,et al.  Role of the ubiquitin‐selective CDC48UFD1/NPL4 chaperone (segregase) in ERAD of OLE1 and other substrates , 2002, The EMBO journal.

[3]  C. Taxis,et al.  Protein dislocation from the ER requires polyubiquitination and the AAA-ATPase Cdc48 , 2002, Nature Cell Biology.

[4]  K. Fröhlich,et al.  AAA-ATPase p97/Cdc48p, a Cytosolic Chaperone Required for Endoplasmic Reticulum-Associated Protein Degradation , 2002, Molecular and Cellular Biology.

[5]  Tom A. Rapoport,et al.  The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol , 2001, Nature.

[6]  R. Hampton,et al.  HRD4/NPL4 is required for the proteasomal processing of ubiquitinated ER proteins. , 2001, Molecular biology of the cell.

[7]  S. Jentsch,et al.  Mobilization of Processed, Membrane-Tethered SPT23 Transcription Factor by CDC48UFD1/NPL4, a Ubiquitin-Selective Chaperone , 2001, Cell.

[8]  M. Maurizi,et al.  AAA proteins: in search of a common molecular basis , 2001, EMBO reports.

[9]  K. Tanaka,et al.  VCP/p97 in abnormal protein aggregates, cytoplasmic vacuoles, and cell death, phenotypes relevant to neurodegeneration , 2001, Cell Death and Differentiation.

[10]  P. Silver,et al.  The conserved npl4 protein complex mediates proteasome-dependent membrane-bound transcription factor activation. , 2001, Molecular biology of the cell.

[11]  Chou-Chi H. Li,et al.  Valosin-containing protein is a multi-ubiquitin chain-targeting factor required in ubiquitin–proteasome degradation , 2001, Nature Cell Biology.

[12]  A. Wilkinson,et al.  AAA+ superfamily ATPases: common structure–diverse function , 2001, Genes to cells : devoted to molecular & cellular mechanisms.

[13]  Vivian X. Fu,et al.  Functional Analysis of the Trypanosomal AAA ProteinTbVCP with trans-Dominant ATP Hydrolysis Mutants* , 2001, The Journal of Biological Chemistry.

[14]  E. Chevet,et al.  Tyrosine phosphorylation of p97 regulates transitional endoplasmic reticulum assembly in vitro. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[15]  M van Heel,et al.  Structure of the AAA ATPase p97. , 2000, Molecular cell.

[16]  I Rouiller,et al.  A major conformational change in p97 AAA ATPase upon ATP binding. , 2000, Molecular cell.

[17]  S. Ruscetti,et al.  Involvement of the Ubiquitin-Proteasome Pathway in the Degradation of Nontyrosine Kinase-Type Cytokine Receptors of IL-9, IL-2, and Erythropoietin1 , 2000, The Journal of Immunology.

[18]  J. Yates,et al.  Proteasomal proteomics: identification of nucleotide-sensitive proteasome-interacting proteins by mass spectrometric analysis of affinity-purified proteasomes. , 2000, Molecular biology of the cell.

[19]  Ronald D. Vale,et al.  Aaa Proteins , 2000, The Journal of cell biology.

[20]  M. Makarow,et al.  The cytoplasmic chaperone hsp104 is required for conformational repair of heat-denatured proteins in the yeast endoplasmic reticulum. , 1999, Molecular biology of the cell.

[21]  O. H. Griffith,et al.  A robotics-based automated assay for inorganic and organic phosphates. , 1999, Analytical biochemistry.

[22]  S. Jentsch,et al.  A Novel Ubiquitination Factor, E4, Is Involved in Multiubiquitin Chain Assembly , 1999, Cell.

[23]  G. Warren,et al.  The p47 co‐factor regulates the ATPase activity of the membrane fusion protein, p97 , 1998, FEBS letters.

[24]  S. Lindquist,et al.  The ATPase Activity of Hsp104, Effects of Environmental Conditions and Mutations* , 1998, The Journal of Biological Chemistry.

[25]  D. Longo,et al.  Involvement of Valosin-containing Protein, an ATPase Co-purified with IκBα and 26 S Proteasome, in Ubiquitin-Proteasome-mediated Degradation of IκBα* , 1998, The Journal of Biological Chemistry.

[26]  H. Zischka,et al.  Tyrosine phosphorylation regulates cell cycle-dependent nuclear localization of Cdc48p. , 1998, Molecular biology of the cell.

[27]  F. Madeo,et al.  Identification of the regions of porcine VCP preventing its function in Saccharomyces cerevisiae. , 1997, Gene.

[28]  M. Purugganan,et al.  The Evolution of the Conserved ATPase Domain (CAD): Reconstructing the History of an Ancient Protein Module , 1997, Journal of Molecular Evolution.

[29]  Reinhard Jahn,et al.  Structure and Conformational Changes in NSF and Its Membrane Receptor Complexes Visualized by Quick-Freeze/Deep-Etch Electron Microscopy , 1997, Cell.

[30]  W. Baumeister,et al.  Cloning, sequencing and expression of VAT, a CDC48/p97 ATPase homologue from the archaeon Thermoplasma acidophilum , 1997, FEBS letters.

[31]  A. Varshavsky,et al.  Cdc48p interacts with Ufd3p, a WD repeat protein required for ubiquitin‐mediated proteolysis in Saccharomyces cerevisiae. , 1996, The EMBO journal.

[32]  A. Bernstein,et al.  Each Domain of the N-Ethylmaleimide-sensitive Fusion Protein Contributes to Its Transport Activity (*) , 1995, The Journal of Biological Chemistry.

[33]  D. Mecke,et al.  The ATPase activity of purified CDC48p from Saccharomyces cerevisiae shows complex dependence on ATP-, ADP-, and NADH-concentrations and is completely inhibited by NEM. , 1995, Biochimica et biophysica acta.

[34]  Susan Lindquist,et al.  Protein disaggregation mediated by heat-shock protein Hspl04 , 1994, Nature.

[35]  Sudheer Kumar Singh,et al.  Mutational analysis demonstrates different functional roles for the two ATP-binding sites in ClpAP protease from Escherichia coli. , 1994, The Journal of biological chemistry.

[36]  J. Rothman,et al.  N-ethylmaleimide-sensitive fusion protein: a trimeric ATPase whose hydrolysis of ATP is required for membrane fusion , 1994, The Journal of cell biology.

[37]  L. Samelson,et al.  Biochemical characterization of valosin-containing protein, a protein tyrosine kinase substrate in hematopoietic cells. , 1994, The Journal of biological chemistry.

[38]  S. Lindquist,et al.  Saccharomyces cerevisiae Hsp104 protein. Purification and characterization of ATP-induced structural changes. , 1994, The Journal of biological chemistry.

[39]  K. Kohn,et al.  Role of the p53 tumor suppressor gene in cell cycle arrest and radiosensitivity of Burkitt's lymphoma cell lines. , 1993, Cancer research.

[40]  J. Bromberg,et al.  Alterations in a yeast protein resembling HIV Tat-binding protein relieve requirement for an acidic activation domain in GAL4 , 1992, Nature.

[41]  J. Harris,et al.  Ubiquitous soluble Mg(2+)-ATPase complex. A structural study. , 1992, Journal of molecular biology.

[42]  D. Botstein,et al.  Yeast cell cycle protein CDC48p shows full-length homology to the mammalian protein VCP and is a member of a protein family involved in secretion, peroxisome formation, and gene expression , 1991, The Journal of cell biology.

[43]  J. Peters,et al.  An abundant and ubiquitous homo‐oligomeric ring‐shaped ATPase particle related to the putative vesicle fusion proteins Sec18p and NSF. , 1990, The EMBO journal.

[44]  J. Rothman,et al.  Purification of an N-ethylmaleimide-sensitive protein catalyzing vesicular transport. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[45]  M. Brownstein,et al.  Use of a cDNA clone to identify a supposed precursor protein containing valosin , 1987, Nature.

[46]  J. Walker,et al.  Distantly related sequences in the alpha‐ and beta‐subunits of ATP synthase, myosin, kinases and other ATP‐requiring enzymes and a common nucleotide binding fold. , 1982, The EMBO journal.

[47]  P. A. Lanzetta,et al.  An improved assay for nanomole amounts of inorganic phosphate. , 1979, Analytical biochemistry.