Proteins are unfolded on the surface of the ATPase ring before transport into the proteasome.

The 19S component of the 26S proteasome contains six ATPase subunits. To clarify how they unfold and translocate proteins into the 20S proteasome for degradation, we studied the homologous archaebacterial proteasome-regulatory ATPase complex PAN and the globular substrate GFP-SsrA. When we attached a small (Biotin) or large (Biotin-Avidin) moiety near its N terminus or a Biotin near its C terminus, GFP-SsrA was unfolded and degraded. However, attaching Avidin near its C terminus blocked passage through PAN and prevented GFP-SsrA degradation. Though not translocated, GFP-Avidin still underwent ATP-dependent unfolding. Moreover, it remained bound to PAN and inhibited further proteolysis. Therefore, (1) translocation and degradation of this substrate require threading through the ATPase in a C to N direction and (2) translocation does not cause but follows ATP-dependent unfolding, which occurs on the surface of the ATPase ring.

[1]  R. Huber,et al.  A gated channel into the proteasome core particle , 2000, Nature Structural Biology.

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

[3]  S. Gottesman,et al.  Regulatory Subunits of Energy-Dependent Proteases , 1997, Cell.

[4]  T. Kunkel.  Rapid and efficient site-specific mutagenesis without phenotypic selection. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[5]  A. Goldberg,et al.  PAN, the proteasome-activating nucleotidase from archaebacteria, is a protein-unfolding molecular chaperone , 2000, Nature Cell Biology.

[6]  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.

[7]  A. Goldberg,et al.  The axial channel of the proteasome core particle is gated by the Rpt2 ATPase and controls both substrate entry and product release. , 2001, Molecular cell.

[8]  Alexei F. Kisselev,et al.  Processive Degradation of Proteins and Other Catalytic Properties of the Proteasome from Thermoplasma acidophilum* , 1997, The Journal of Biological Chemistry.

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

[10]  R. Kopito,et al.  Impairment of the ubiquitin-proteasome system by protein aggregation. , 2001, Science.

[11]  H. Saibil,et al.  Molecular chaperones: containers and surfaces for folding, stabilising or unfolding proteins. , 2000, Current opinion in structural biology.

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

[13]  K Tanaka,et al.  Structure and functions of the 20S and 26S proteasomes. , 1996, Annual review of biochemistry.

[14]  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.

[15]  Alexei F. Kisselev,et al.  Range of Sizes of Peptide Products Generated during Degradation of Different Proteins by Archaeal Proteasomes* , 1998, The Journal of Biological Chemistry.

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

[17]  A. Matouschek,et al.  ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal. , 2001, Molecular cell.

[18]  Wolfgang Baumeister,et al.  The Proteasome: Paradigm of a Self-Compartmentalizing Protease , 1998, Cell.

[19]  E. Strickland,et al.  Recognition of Misfolding Proteins by PA700, the Regulatory Subcomplex of the 26 S Proteasome* , 2000, The Journal of Biological Chemistry.

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

[21]  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.

[22]  J. Vieira,et al.  A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. , 1982, Gene.

[23]  Wolfgang Hilt,et al.  Proteasomes: The World of Regulatory Proteolysis , 2000 .

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

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

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

[27]  C. Larsen,et al.  Protein Translocation Channels in the Proteasome and Other Proteases , 1997, Cell.

[28]  A. Goldberg,et al.  The ubiquitin-proteasome pathway is required for processing the NF-kappa B1 precursor protein and the activation of NF-kappa B. , 1994, Cell.

[29]  W. Baumeister,et al.  The 26S proteasome: a molecular machine designed for controlled proteolysis. , 1999, Annual review of biochemistry.

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

[31]  A. Goldberg,et al.  Cellular Defenses against Unfolded Proteins A Cell Biologist Thinks about Neurodegenerative Diseases , 2001, Neuron.

[32]  A. Goldberg,et al.  An Archaebacterial ATPase, Homologous to ATPases in the Eukaryotic 26 S Proteasome, Activates Protein Breakdown by 20 S Proteasomes* , 1999, The Journal of Biological Chemistry.

[33]  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.