The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system.

Interruption of translation in Escherichia coli can lead to the addition of an 11-residue carboxy-terminal peptide tail to the nascent chain. This modification is mediated by SsrA RNA (also called 10Sa RNA and tmRNA) and marks the tagged polypeptide for proteolysis. Degradation in vivo of lambda repressor amino-terminal domain variants bearing this carboxy-terminal SsrA peptide tag is shown here to depend on the cytoplasmic proteases ClpXP and ClpAP. Degradation in vitro of SsrA-tagged substrates was reproduced with purified components and required a substrate with a wild-type SsrA tail, the presence of both ClpP and either ClpA or ClpX, and ATP. Clp-dependent proteolysis accounts for most degradation of SsrA-tagged amino-domain substrates at 32 degrees C, but additional proteases contribute to the degradation of some of these SsrA-tagged substrates at 39 degrees C. The existence of multiple cytoplasmic proteases that function in SsrA quality-control surveillance suggests that the SsrA tag is designed to serve as a relatively promiscuous signal for proteolysis. Having diverse degradation systems able to recognize this tag may increase degradation capacity, permit degradation of a wide variety of different tagged proteins, or allow SsrA-tagged proteins to be degraded under different growth conditions.

[1]  P. Bouloc,et al.  Cell growth and lambda phage development controlled by the same essential Escherichia coli gene, ftsH/hflB. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

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

[3]  S. Gottesman,et al.  Role of the Heat Shock Protein DnaJ in the Lon-dependent Degradation of Naturally Unstable Proteins* , 1996, The Journal of Biological Chemistry.

[4]  H. Yanagi,et al.  Synergistic roles of HslVU and other ATP-dependent proteases in controlling in vivo turnover of sigma32 and abnormal proteins in Escherichia coli , 1997, Journal of bacteriology.

[5]  T. Baker,et al.  PDZ-like Domains Mediate Binding Specificity in the Clp/Hsp100 Family of Chaperones and Protease Regulatory Subunits , 1997, Cell.

[6]  S. Gottesman,et al.  Excision of a P4-like cryptic prophage leads to Alp protease expression in Escherichia coli , 1994, Journal of bacteriology.

[7]  D. Court,et al.  The relationship between function and DNA sequence in an intercistronic regulatory region in phage λ , 1978, Nature.

[8]  A. Steven,et al.  Enzymatic and Structural Similarities between theEscherichia coli ATP-dependent Proteases, ClpXP and ClpAP* , 1998, The Journal of Biological Chemistry.

[9]  Jordan,et al.  Structure of the lambda complex at 2.5 A resolution: details of the repressor-operator interactions , 1988, Science.

[10]  J U Bowie,et al.  Identification of C-terminal extensions that protect proteins from intracellular proteolysis. , 1989, The Journal of biological chemistry.

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

[12]  J. F. Atkins,et al.  Probing the structure of the Escherichia coli 10Sa RNA (tmRNA). , 1997, RNA.

[13]  S. Gottesman,et al.  A molecular chaperone, ClpA, functions like DnaK and DnaJ. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[14]  C. Georgopoulos,et al.  Isolation and characterization of ClpX, a new ATP-dependent specificity component of the Clp protease of Escherichia coli. , 1993, The Journal of biological chemistry.

[15]  S. Gottesman,et al.  Regulation of capsular polysaccharide synthesis in Escherichia coli K-12: characterization of three regulatory genes , 1985, Journal of bacteriology.

[16]  D. Bartel,et al.  Phylogenetic analysis of tmRNA secondary structure. , 1996, RNA.

[17]  K. Biemann,et al.  C‐terminal specific protein degradation: Activity and substrate specificity of the Tsp protease , 1995, Protein science : a publication of the Protein Society.

[18]  S. Gottesman,et al.  Protein quality control: triage by chaperones and proteases. , 1997, Genes & development.

[19]  R. Moritz,et al.  C-terminal Extension of Truncated Recombinant Proteins in Escherichia coli with a 10Sa RNA Decapeptide(*) , 1995, The Journal of Biological Chemistry.

[20]  Mark Ptashne,et al.  Regulatory functions of the λ repressor reside in the amino-terminal domain , 1979, Nature.

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

[22]  T. Baker,et al.  Disassembly of the Mu transposase tetramer by the ClpX chaperone. , 1995, Genes & development.

[23]  A. Toussaint,et al.  C-terminal deletions can suppress temperature-sensitive mutations and change dominance in the phage Mu repressor. , 1996, Genetics.

[24]  S. Gottesman,et al.  The ATP-dependent Clp protease of Escherichia coli. Sequence of clpA and identification of a Clp-specific substrate. , 1990, The Journal of biological chemistry.

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

[26]  C. Squires,et al.  The Clp proteins: proteolysis regulators or molecular chaperones? , 1992, Journal of bacteriology.

[27]  D. Friedman,et al.  A role for a small stable RNA in modulating the activity of DNA-binding proteins , 1995, Cell.

[28]  S. Gottesman,et al.  Proteases and their targets in Escherichia coli. , 1996, Annual review of genetics.

[29]  H. Inokuchi,et al.  A tRNA-like structure is present in 10Sa RNA, a small stable RNA from Escherichia coli. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[30]  H. Mori,et al.  Escherichia coli FtsH is a membrane‐bound, ATP‐dependent protease which degrades the heat‐shock transcription factor sigma 32. , 1995, The EMBO journal.

[31]  M. Khattar.  Overexpression of the hslVU operon suppresses SOS‐mediated inhibition of cell division in Escherichia coli , 1997, FEBS letters.

[32]  C. Georgopoulos,et al.  Identification and characterization of HsIV HsIU (ClpQ ClpY) proteins involved in overall proteolysis of misfolded proteins in Escherichia coli. , 1996, The EMBO journal.

[33]  H. Goldberg,et al.  The DNA binding arm of lambda repressor: critical contacts from a flexible region. , 1991, Science.

[34]  D. Friedman,et al.  Role for 10Sa RNA in the growth of lambda-P22 hybrid phage , 1994, Journal of bacteriology.

[35]  S. Gottesman,et al.  Insertional mutagenesis of the lon gene in Escherichia coli: lon is dispensable , 1985, Journal of bacteriology.

[36]  B L Trus,et al.  Homology in structural organization between E. coli ClpAP protease and the eukaryotic 26 S proteasome. , 1995, Journal of molecular biology.

[37]  P. Bouloc,et al.  Degradation of sigma 32, the heat shock regulator in Escherichia coli, is governed by HflB. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[38]  W. Sly,et al.  Mechanism of λc17cI virulence , 1969 .

[39]  A. Toussaint,et al.  Bacteriophage Mu repressor as a target for the Escherichia coli ATP‐dependent Clp Protease. , 1996, The EMBO journal.

[40]  S. Gottesman,et al.  Alp suppression of Lon: dependence on the slpA gene , 1994, Journal of bacteriology.

[41]  R. Sauer,et al.  Folded proteins occur frequently in libraries of random amino acid sequences. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[42]  S. Gottesman,et al.  ClpX, an alternative subunit for the ATP-dependent Clp protease of Escherichia coli. Sequence and in vivo activities. , 1993, The Journal of biological chemistry.

[43]  R. Sauer,et al.  Tsp: a tail-specific protease that selectively degrades proteins with nonpolar C termini. , 1992, Proceedings of the National Academy of Sciences of the United States of America.