ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal.

Protein unfolding is a key step in several cellular processes, including protein translocation across some membranes and protein degradation by ATP-dependent proteases. ClpAP protease and the proteasome can actively unfold proteins in a process that hydrolyzes ATP. Here we show that these proteases seem to catalyze unfolding by processively unraveling their substrates from the attachment point of the degradation signal. As a consequence, the ability of a protein to be degraded depends on its structure as well as its stability. In multidomain proteins, independently stable domains are unfolded sequentially. We show that these results can explain the limited degradation by the proteasome that occurs in the processing of the precursor of the transcription factor NF-kappaB.

[1]  A. Matouschek,et al.  Active unfolding of precursor proteins during mitochondrial protein import , 1997, The EMBO journal.

[2]  W. Greene,et al.  Cotranslational dimerization of the Rel homology domain of NF‐κB1 generates p50–p105 heterodimers and is required for effective p50 production , 2000, The EMBO journal.

[3]  A. Horwich,et al.  ClpA mediates directional translocation of substrate proteins into the ClpP protease , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[4]  W. Baumeister,et al.  26S proteasome structure revealed by three-dimensional electron microscopy. , 1998, Journal of structural biology.

[5]  G. Nabel,et al.  Differential regulation of NF-kappaB2(p100) processing and control by amino-terminal sequences , 1996, Molecular and cellular biology.

[6]  R. Sauer,et al.  Role of a Peptide Tagging System in Degradation of Proteins Synthesized from Damaged Messenger RNA , 1996, Science.

[7]  A. Fersht,et al.  Contribution of hydrophobic interactions to protein stability , 1988, Nature.

[8]  R. Sauer,et al.  Carboxy-terminal determinants of intracellular protein degradation. , 1990, Genes & development.

[9]  A. Matouschek,et al.  Mitochondria unfold precursor proteins by unraveling them from their N-termini , 1999, Nature Structural Biology.

[10]  A. Steven,et al.  Visualization of substrate binding and translocation by the ATP-dependent protease, ClpXP. , 2000, Molecular cell.

[11]  S. Harrison,et al.  Structure of an IκBα/NF-κB Complex , 1998, Cell.

[12]  T. Endo,et al.  Latent membrane perturbation activity of a mitochondrial precursor protein is exposed by unfolding. , 1988, The EMBO journal.

[13]  T. Ahrens,et al.  Water storage in the mantle , 1989, Nature.

[14]  Tsutomu Nakamura,et al.  Systematic circular permutation of an entire protein reveals essential folding elements , 2000, Nature Structural Biology.

[15]  A. Steven,et al.  Translocation pathway of protein substrates in ClpAP protease , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[16]  W. Baumeister,et al.  Conformational constraints in protein degradation by the 20S proteasome , 1995, Nature Structural Biology.

[17]  R. Huber,et al.  Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 A resolution. , 1995, Science.

[18]  C. Branden,et al.  Introduction to protein structure , 1991 .

[19]  Y. Ishii,et al.  Functional dissection of a cell-division inhibitor, SulA, of Escherichia coli and its negative regulation by Lon , 1997, Molecular and General Genetics MGG.

[20]  A. Varshavsky,et al.  The degradation signal in a short-lived protein , 1989, Cell.

[21]  T. Ueda,et al.  Unfolding rates of globular proteins determined by kinetics of proteolysis. , 1986, Journal of molecular biology.

[22]  A. Fersht,et al.  A calorimetric study of the thermal stability of barstar and its interaction with barnase. , 1995, Biochemistry.

[23]  J. Tobias,et al.  Universality and structure of the N-end rule. , 1989, The Journal of biological chemistry.

[24]  J. Hoskins,et al.  Unfolding and internalization of proteins by the ATP-dependent proteases ClpXP and ClpAP. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Tom Maniatis,et al.  The ubiquitinproteasome pathway is required for processing the NF-κB1 precursor protein and the activation of NF-κB , 1994, Cell.

[26]  Erica S. Johnson,et al.  Methotrexate Inhibits Proteolysis of Dihydrofolate Reductase by the N-end Rule Pathway (*) , 1995, The Journal of Biological Chemistry.

[27]  S. Jentsch,et al.  Activation of a Membrane-Bound Transcription Factor by Regulated Ubiquitin/Proteasome-Dependent Processing , 2000, Cell.

[28]  R. Sauer,et al.  The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system. , 1998, Genes & development.

[29]  A. Horwich,et al.  Global unfolding of a substrate protein by the Hsp100 chaperone ClpA , 1999, Nature.

[30]  P. Kloetzel,et al.  The base of the proteasome regulatory particle exhibits chaperone-like activity , 1999, Nature Cell Biology.

[31]  Walter Neupert,et al.  The YTA10–12 Complex, an AAA Protease with Chaperone-like Activity in the Inner Membrane of Mitochondria , 1996, Cell.

[32]  T. Baker,et al.  Dynamics of substrate denaturation and translocation by the ClpXP degradation machine. , 2000, Molecular cell.

[33]  Christine B. Trame,et al.  Crystal and Solution Structures of an HslUV Protease–Chaperone Complex , 2000, Cell.

[34]  G. Ghosh,et al.  The Crystal Structure of the IκBα/NF-κB Complex Reveals Mechanisms of NF-κB Inactivation , 1998, Cell.

[35]  A. Ciechanover,et al.  Ubiquitin-mediated processing of NF-kappa B transcriptional activator precursor p105. Reconstitution of a cell-free system and identification of the ubiquitin-carrier protein, E2, and a novel ubiquitin-protein ligase, E3, involved in conjugation. , 1995, The Journal of biological chemistry.

[36]  A. Matouschek,et al.  Effect of the protein import machinery at the mitochondrial surface on precursor stability. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[37]  A. C. Chang,et al.  Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid , 1978, Journal of bacteriology.

[38]  M. Kessel,et al.  Molecular properties of ClpAP protease of Escherichia coli: ATP-dependent association of ClpA and clpP. , 1998, Biochemistry.

[39]  H. Stahlberg,et al.  Mitochondrial Lon of Saccharomyces cerevisiae is a ring-shaped protease with seven flexible subunits. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[40]  W. Welch,et al.  Neurodegeneration: Chaperoning brain diseases , 1998, Nature.

[41]  R. Hartley.  A two-state conformational transition of the extracellular ribonuclease of Bacillus amyloliquefaciens (barnase) induced by sodium dodecyl sulfate. , 1975, Biochemistry.

[42]  A. Varshavsky.  The N-end rule , 1992, Cell.

[43]  J. Tobias,et al.  The N-end rule in bacteria. , 1991, Science.

[44]  A. Hershko,et al.  Specificity of binding of NH2-terminal residue of proteins to ubiquitin-protein ligase. Use of amino acid derivatives to characterize specific binding sites. , 1988, The Journal of biological chemistry.

[45]  A. Horwich,et al.  Chaperone rings in protein folding and degradation. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[46]  Stanley N Cohen,et al.  Phenotypic expression in E. coli of a DNA sequence coding for mouse dihydrofolate reductase , 1978, Nature.

[47]  J. Rood,et al.  Cloning of the Escherichia coli K-12 dihydrofolate reductase gene following mu-mediated transposition. , 1980, Gene.

[48]  M. Eilers,et al.  Binding of a specific ligand inhibits import of a purified precursor protein into mitochondria , 1986, Nature.

[49]  P. Bouloc,et al.  Degradation of carboxy-terminal-tagged cytoplasmic proteins by the Escherichia coli protease HflB (FtsH). , 1998, Genes & development.

[50]  J. Hoskins,et al.  Protein binding and unfolding by the chaperone ClpA and degradation by the protease ClpAP. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[51]  S. Gottesman,et al.  ATP-dependent Degradation of CcdA by Lon Protease , 1996, The Journal of Biological Chemistry.

[52]  Jimin Wang,et al.  The Structure of ClpP at 2.3 Å Resolution Suggests a Model for ATP-Dependent Proteolysis , 1997, Cell.

[53]  L. Grivell,et al.  Promotion of Mitochondrial Membrane Complex Assembly by a Proteolytically Inactive Yeast Lon , 1996, Science.

[54]  R A Sayle,et al.  RASMOL: biomolecular graphics for all. , 1995, Trends in biochemical sciences.

[55]  A. Varshavsky,et al.  In vivo half-life of a protein is a function of its amino-terminal residue. , 1986, Science.

[56]  G. Lorimer,et al.  Complex interactions between the chaperonin 60 molecular chaperone and dihydrofolate reductase. , 1991, Biochemistry.

[57]  M. Hochstrasser.  Ubiquitin-dependent protein degradation. , 1996, Annual review of genetics.

[58]  A. Fersht,et al.  Mapping the transition state and pathway of protein folding by protein engineering , 1989, Nature.

[59]  G. Struhl,et al.  Regulation of the Hedgehog and Wingless signalling pathways by the F-box/WD40-repeat protein Slimb , 1998, Nature.

[60]  R. Huber,et al.  Structure of 20S proteasome from yeast at 2.4Å resolution , 1997, Nature.

[61]  W. Greene,et al.  Cotranslational Biogenesis of NF-κB p50 by the 26S Proteasome , 1998, Cell.