Tricorn protease - The core of a modular proteolytic system

Science

Volumn 274, Issue 5291, 1996, Pages 1385-1389

  Tamura, Tomohiro ^a   Tamura, Noriko ^a   Cejka, Zdenka ^a   Hegerl, Reiner ^a   Lottspeich, Friedrich ^a   Baumeister, Wolfgang ^a  

^a MAX PLANCK INSTITUTE OF BIOCHEMISTRY   (Germany)

Author keywords

[No Author keywords available]

Indexed keywords

BACTERIAL DNA; BACTERIAL ENZYME; CHYMOTRYPSIN; HYDROLASE; PROTEASOME; PROTEINASE; TRYPSIN;

AMINO ACID SEQUENCE; ARTICLE; CATALYSIS; CONTROLLED STUDY; ENZYME ACTIVITY; ENZYME PURIFICATION; NONHUMAN; OPEN READING FRAME; POLYACRYLAMIDE GEL ELECTROPHORESIS; POLYMERASE CHAIN REACTION; PRIORITY JOURNAL; PROTEIN DEGRADATION; THERMOPLASMA;

AMINO ACID SEQUENCE; CLONING, MOLECULAR; CYSTEINE ENDOPEPTIDASES; ENDOPEPTIDASES; GENES, BACTERIAL; MICROSCOPY, ELECTRON; MODELS, MOLECULAR; MOLECULAR SEQUENCE DATA; MOLECULAR WEIGHT; MULTIENZYME COMPLEXES; PEPTIDES; PROTEASOME ENDOPEPTIDASE COMPLEX; PROTEIN CONFORMATION; PROTEIN FOLDING; PROTEIN STRUCTURE, TERTIARY; RECOMBINANT PROTEINS; SUBSTRATE SPECIFICITY; THERMOPLASMA;

ARCHAEA; BACTERIA (MICROORGANISMS); MIRIDAE; THERMOPLASMA;

EID: 0029831775     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.274.5291.1385     Document Type: Article

References (33)

1. J. Löwe et al., Science 268, 533 (1995); E. Seemüller et al., ibid., P. 579; G. pühler et al., EMBO J. 11, 1607 (1992); T. Wenzel and W. Baumeister, Nature Struct. Biol. 2, 199 (1995); A. Lupas, A. J. Koster, W. Baumeister, Enzyme Protein 47, 252 (1993); D. M. Rubin and D. Finley, Curr. Biol. 5, 854 (1995).
2. J. Löwe et al., Science 268, 533 (1995); E. Seemüller et al., ibid., P. 579; G. pühler et al., EMBO J. 11, 1607 (1992); T. Wenzel and W. Baumeister, Nature Struct. Biol. 2, 199 (1995); A. Lupas, A. J. Koster, W. Baumeister, Enzyme Protein 47, 252 (1993); D. M. Rubin and D. Finley, Curr. Biol. 5, 854 (1995).
3. J. Löwe et al., Science 268, 533 (1995); E. Seemüller et al., ibid., P. 579; G. pühler et al., EMBO J. 11, 1607 (1992); T. Wenzel and W. Baumeister, Nature Struct. Biol. 2, 199 (1995); A. Lupas, A. J. Koster, W. Baumeister, Enzyme Protein 47, 252 (1993); D. M. Rubin and D. Finley, Curr. Biol. 5, 854 (1995).
4. J. Löwe et al., Science 268, 533 (1995); E. Seemüller et al., ibid., P. 579; G. pühler et al., EMBO J. 11, 1607 (1992); T. Wenzel and W. Baumeister, Nature Struct. Biol. 2, 199 (1995); A. Lupas, A. J. Koster, W. Baumeister, Enzyme Protein 47, 252 (1993); D. M. Rubin and D. Finley, Curr. Biol. 5, 854 (1995).
5. J. Löwe et al., Science 268, 533 (1995); E. Seemüller et al., ibid., P. 579; G. pühler et al., EMBO J. 11, 1607 (1992); T. Wenzel and W. Baumeister, Nature Struct. Biol. 2, 199 (1995); A. Lupas, A. J. Koster, W. Baumeister, Enzyme Protein 47, 252 (1993); D. M. Rubin and D. Finley, Curr. Biol. 5, 854 (1995).
6. J. Löwe et al., Science 268, 533 (1995); E. Seemüller et al., ibid., P. 579; G. pühler et al., EMBO J. 11, 1607 (1992); T. Wenzel and W. Baumeister, Nature Struct. Biol. 2, 199 (1995); A. Lupas, A. J. Koster, W. Baumeister, Enzyme Protein 47, 252 (1993); D. M. Rubin and D. Finley, Curr. Biol. 5, 854 (1995).
7. B. Dahlmann et al. FEBS Lett. 251, 125 (1989); J. A. Maupin-Furlow and J. G. Ferry, J. Biol. Chem. 270, 28617 (1995).
8. B. Dahlmann et al. FEBS Lett. 251, 125 (1989); J. A. Maupin-Furlow and J. G. Ferry, J. Biol. Chem. 270, 28617 (1995).
9. A. Lupas, P. Zwickl, W. Baumeister, Trends Biochem. Sci. 19, 533 (1994).
10. T. Tamura et al., Curr. Biol. 5, 766 (1995).
11. M. Orlowski, Biochemistry 29, 10289 (1990); K. Tanaka, T. Tamura, T. Yoshimura, A. Ichihara, New Biol. 4, 173 (1992); A. J. Rivett, Biochem. J. 291, 1 (1993).
12. M. Orlowski, Biochemistry 29, 10289 (1990); K. Tanaka, T. Tamura, T. Yoshimura, A. Ichihara, New Biol. 4, 173 (1992); A. J. Rivett, Biochem. J. 291, 1 (1993).
13. M. Orlowski, Biochemistry 29, 10289 (1990); K. Tanaka, T. Tamura, T. Yoshimura, A. Ichihara, New Biol. 4, 173 (1992); A. J. Rivett, Biochem. J. 291, 1 (1993).
14. O. Coux, K. Tanaka, A. L. Goldberg, Annu. Rev. Biochem. 65, 801 (1996).
15. 2, 5 mM ATP, and DNasell (10 μg/ml)] and incubated for 30 min at room temperature before lysis by sonication. The lysate was centrifuged for 1 hour at 61,700g. Glycerol was added to the supernatant (the final 20%), which was stored at -80°C until use. Throughout the purification procedures for TRI and F1 protein, peptidase activity was tested by addition of either pooled LMW fractions or pooled HMW fractions, which were fractionated by a 10 to 40% glycerol density gradient by means of centrifugation at 25,000 rpm for 22 hours at 4°C with a SW28 rotor (Beckman). For the F2 protein purification, the peptidase activities were tested by addition of recombinant TRI (rTRI). Peptidase assays were performed in 100 μl of 50 mM tris-HCl (pH 8.0), and released AMC was measured as previously described (4). For isolation of TRI, 2.8 g of lysate from T. acidophilum cells were used for purification. The purified protein (900 μg) was dialyzed against 50 mM tris-HCl (pH 7.5) with 20% glycerol and stored at 4°C. For isolation of the F1 protein, 900 mg of lysate from T. acidophilum cells was subjected to the purification procedure. The purified protein (40 μg) was dialyzed with 25 mM tris-HCl buffer (pH 7.5) containing 1 mM DTT, and was stored at 4°C, For isolation of F2, 1.36 g of lysate from T. acidophilum cells were used for purification. Partially purified F2 (390 μg) was dialyzed with 50 mM tris-HCl buffer (pH 7.5) and stored at 4°C. Purified TRI and the F1 protein were subjected to amino acid sequence analysis. A detailed description of the purification procedure is available on request (e-mail: tamura@vms.biochem. mpg.de).
16. 2-terminal truncated TRI-encoding gene. To obtain the missing region, chromosomal DNA was digested by Ava I or Ban II, circularized with T4 ligase, and subjected to inverse PCR. The amplified 1317-bp and 2079-bp fragments were purified and subjected to sequence analyses. The cloned ORF encoding the 120-kD protein has two possible initiation codons, Met1 and Met5; and a putative ribosomal binding site, which is found downstream of the initiation codon in archaea [W. Zillig et al., Eur. J. Biochem. 173, 473 (1988)], is located between both methionines. We tentatively selected Met1 as the initiation codon because it encodes the longest ORF.
17. 2-terminal truncated TRI-encoding gene. To obtain the missing region, chromosomal DNA was digested by Ava I or Ban II, circularized with T4 ligase, and subjected to inverse PCR. The amplified 1317-bp and 2079-bp fragments were purified and subjected to sequence analyses. The cloned ORF encoding the 120-kD protein has two possible initiation codons, Met1 and Met5; and a putative ribosomal binding site, which is found downstream of the initiation codon in archaea [W. Zillig et al., Eur. J. Biochem. 173, 473 (1988)], is located between both methionines. We tentatively selected Met1 as the initiation codon because it encodes the longest ORF.
18. 2-terminal truncated TRI-encoding gene. To obtain the missing region, chromosomal DNA was digested by Ava I or Ban II, circularized with T4 ligase, and subjected to inverse PCR. The amplified 1317-bp and 2079-bp fragments were purified and subjected to sequence analyses. The cloned ORF encoding the 120-kD protein has two possible initiation codons, Met1 and Met5; and a putative ribosomal binding site, which is found downstream of the initiation codon in archaea [W. Zillig et al., Eur. J. Biochem. 173, 473 (1988)], is located between both methionines. We tentatively selected Met1 as the initiation codon because it encodes the longest ORF.
19. G. I. H. Liou, L. Geng, W. Baehr, in The Molecular Biology of the Retina: Basic and Clinically Relevant Studies, D. B. Farber and G. J. Chader, Eds. (Wiley-Liss, New York, 1991), pp. 115-137.
20. S. Jentsch, Science 271, 955 (1996); K. C. Keiler, P. R. H. Waller, R. T. Sauer, ibid., p. 990.
21. S. Jentsch, Science 271, 955 (1996); K. C. Keiler, P. R. H. Waller, R. T. Sauer, ibid., p. 990.
22. K. C. Keiler and R. T. Sauer, J. Biol. Chem. 270, 28864 (1995).
23. 6-tagged TRI was expressed in E. coli BL21(DE3) cells [F. W. Studier, A. H. Rosenberg, J. J. Dunn, J. W. Dubendorff, Methods Enzymol. 185, 60 (1990)] containing a pUBS520 plasmid encoding the dnaY gene [U. Brinkmann, R. E. Mattes, P. Buckel, Gene 85, 109 (1989)]. The recombinant proteins were purified on a Ni-NTA resin (Diagen, Hilden, Germany) and a Mono-Q column and dialyzed against 50 mM tris-HCl (pH 7.5) with 20% glycerol. The expression level of rTRI in pUBS520-transformed BL21 cells was 10 times higher than in normal BL21 cells. The structure of recombinant protein was confirmed by electron microscopy.
24. 6-tagged TRI was expressed in E. coli BL21(DE3) cells [F. W. Studier, A. H. Rosenberg, J. J. Dunn, J. W. Dubendorff, Methods Enzymol. 185, 60 (1990)] containing a pUBS520 plasmid encoding the dnaY gene [U. Brinkmann, R. E. Mattes, P. Buckel, Gene 85, 109 (1989)]. The recombinant proteins were purified on a Ni-NTA resin (Diagen, Hilden, Germany) and a Mono-Q column and dialyzed against 50 mM tris-HCl (pH 7.5) with 20% glycerol. The expression level of rTRI in pUBS520-transformed BL21 cells was 10 times higher than in normal BL21 cells. The structure of recombinant protein was confirmed by electron microscopy.
25. 6-tagged TRI was expressed in E. coli BL21(DE3) cells [F. W. Studier, A. H. Rosenberg, J. J. Dunn, J. W. Dubendorff, Methods Enzymol. 185, 60 (1990)] containing a pUBS520 plasmid encoding the dnaY gene [U. Brinkmann, R. E. Mattes, P. Buckel, Gene 85, 109 (1989)]. The recombinant proteins were purified on a Ni-NTA resin (Diagen, Hilden, Germany) and a Mono-Q column and dialyzed against 50 mM tris-HCl (pH 7.5) with 20% glycerol. The expression level of rTRI in pUBS520-transformed BL21 cells was 10 times higher than in normal BL21 cells. The structure of recombinant protein was confirmed by electron microscopy.
26. T. Tamura, N. Tamura, W. Baumeister, unpublished results.
27. 3 is assumed.
28. 3 is assumed.
29. 3 is assumed.
30. L. Joshua-Tor, H. E. Xu, S.A. Johnston, D. C. Rees, Science 269, 945 (1995).
31. T. Tamura, N. Tamura, F. Lottspeich, W. Baumeister, FEBS Lett., in press
32. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
33. We thank R. M. Glaeser (University of California, Berkeley). A. Lupas, and M. A. Kania for critically reading the manuscript; M. Boicu for DNA sequencing; and R. Mattes (University of Stuttgart) for providing the dnaY gene. T.T. acknowledges a Postdoctoral Fellowship for Research Abroad from the Japan Society for the Promotion of Science. This work was also supported by a grant from the Human Frontier Science Program to W.B.