Identification and characterization of HsIV HsIU (ClpQ ClpY) proteins involved in overall proteolysis of misfolded proteins in Escherichia coli.

Abstract

Heat shock response in Escherichia coli is autoregulated. Consistent with this, mutations in certain heat shock genes, such as dnaK, dnaJ, grpE or htrC lead to a higher constitutive heat shock gene expression at low temperatures. A similar situation occurs upon accumulation of newly synthesized peptides released prematurely from the ribosomes by puromycin. We looked for gene(s) which, when present in multicopy, prevent the constitutive heat shock response associated with htrC mutant bacteria or caused by the presence of puromycin. One such locus was identified and shown to carry the recently sequenced hslV hslU (clpQ clpY) operon. HslV/ClpQ shares a very high degree of homology with members of the beta‐type subunit, constituting the catalytic core of the 20S proteasome. HslU/ClpY is 50% identical to the ClpX protein of E. coli, which is known to present large polypeptides to its partner, the ATP‐independent proteolytic enzyme ClpP. We show that, in vivo, HslV and HslU interact and participate in the degradation of abnormal puromycylpolypeptides. Biochemical evidence suggests that HslV/ClpQ is an efficient peptidase whose activity is enhanced by HslU/CIpY in the presence of ATP.

Clp Protease Complexes from Photosynthetic and Non-photosynthetic Plastids and Mitochondria of Plants, Their Predicted Three-dimensional Structures, and Functional Implications

“…For nano-LC-ESI-MS/MS, a quadrupole-time of flight (micromass) was used. Separation of peptide mixtures was achieved by using a -Guard PreColumn (LC Packings: inner diameter 300 m, 1 mm long) followed by a PepMap C 18 Reverse Phase Nano Column (LC Packings: inner diameter 180 m, 15 cm long). Peptides were eluted by running a gradient 1 The abbreviations used are: r.m.s.d., root mean square deviation; BN-PAGE, blue native-PAGE; CN-PAGE, colorless native-PAGE; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS, mass spectrometry; nano-LC-ESI-MS/MS, nano-liquid chromatography electrospray ionization-tandem mass spectrometry; NIEF, native isoelectric focusing; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; Tricine, N- [2- Criteria for positive identification of proteins by peptide mass finger printing were at least 5 matching unique peptides (with mass accuracy Ͻ50 ppm, but typically Ͻ10 ppm), allowing no missed cleavage or modifications, and requiring at least 15% coverage of the predicted protein sequence.…”

“…However, E. coli ClpXP can accommodate 2 or 3 polypeptide chains at the same time, leading to an estimation of the opening of 20 -25 Å wide (17). Clp complexes in bacteria can also be formed by ClpQ,Y (HslV and HsIU) proteins, which are unrelated to ClpP (18). No ClpQ,Y homologues has been found in any plant species.…”

Binding of MG132 or Deletion of the Thr Active Sites in HslV Subunits Increases the Affinity of HslV Protease for HslU ATPase and Makes This Interaction Nucleotide-independent

“…HslVU is a two-component ATP-dependent protease in bacteria that comprises HslV protease and HslU ATPase (1)(2)(3)(4)(5). HslV, a homolog of the ␤-subunit of 20 S proteasome, is a selfcompartmentalized protease that has two stacked hexameric rings of identical subunits, each of which has an N-terminal Thr (Thr-1) active site for proteolysis (6 -12).…”

Inhibition of Proteasome Activity Induces Concerted Expression of Proteasome Genes and de Novo Formation of Mammalian Proteasomes

“…Thus, genes encoding Clp proteases in Gram-positive bacteria are negatively controlled by a repressor that is degraded by the ClpCP protease under inducing conditions resulting in a positive autoregulatory feedback loop (18). In Escherichia coli, such a mechanism of self-control was shown for 32 activating gene expression of Clp proteases (19,20).…”