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The ATP-dependent HslVU protease from Escherichia coli is a four-ring structure resembling the proteasome

Nature Structural Biology volume 4, pages 133–139 (1997)Cite this article

Abstract

HslVU is a new two-component protease in Escherichia coli composed of the proteasome-related peptidase HslV and the ATPase HslU. We have used electron microscopy and image analysis to examine the structural organization of HslV and HslU homo-oligomers and the active HslVU enzyme. Electron micrographs of HslV reveal ring-shaped particles, and averaging of top views reveal six-fold rotational symmetry, in contrast to other β-type proteasome subunits, which form rings with seven-fold symmetry. Side views of HslV show two rings stacked together; thus, HslV behaves as dodecamer. The ATPase HslU forms ring-shaped particles in the presence of ATP, AMP-PNP or ADP, suggesting that nucleotide binding, but not hydrolysis, is required for oligomerization. Subunit crosslinking, STEM mass estimation, and analysis of HslU top views indicate that HsIU exists both as hexameric and heptameric rings. With AMP-PNP present, maximal proteolytic activity is observed with a molar ratio of HsIU to HsIV subunits of 1:1, and negative staining electron microscopy shows that HsIV and HsIU form cylindrical four-ring structures in which the HslV dodecamer is flanked at each end by a HslU ring.

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References

  1. Parsell, D.A. & Lindquist, S. The function of heat-shock proteins in stress tolerance: degradation and reactivation of damaged proteins. Ann. Rev. Genet. 27, 437–496 (1993).

    Article  CAS  PubMed  Google Scholar 

  2. Goldberg, A.L. The mechanism and functions of ATP-dependent proteases in bacterial and animal cells. Eur. J. Biochem 203, 9–923 (1992).

    Article  CAS  PubMed  Google Scholar 

  3. Hayes, S.A. & Dice, J.F. Roles of molecular chaperones in protein degradation. J. Cell Biol. 132, 255–258 (1996).

    Article  CAS  PubMed  Google Scholar 

  4. Goff, S.A., Casson, L.P. & Goldberg, A.L. Heat shock regulatory gene htpR influences rates of protein degradation and expression of the Ion gene in Escherichia coli. Proc.Natl.Acad.Sci. USA 81, 6647–6651 (1984).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kroh, H.E. & Simon, L.D. CIpP component of Clp protease is the sigma 32-dependent heat shock protein F21.5. J. Bacteriol. 172, 6026–6034 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Wojtkowiak, D., Georgopoulos, C. & Zylicz, M. Isolation and characterization of CIpX, a new ATP-dependent specificity component of the Clp protease of Escherichia coli. J. Biol. Chem. 268, 22609–22617 (1993).

    CAS  PubMed  Google Scholar 

  7. Tomoyasu, T. et al. Escherichia coll FtsH is a membrane-bound, ATP-dependent protease which degrades the heat-shock transcription factor sigma 32. EMBO J. 14, 2551–2560 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Rohrwild, M. et al. HsIV-HsIU- a novel ATP-dependent protease complex in Escherichia coli related to the eukaryotic proteasome. Proc. Natl. Acad. Sci. USA 93, 5808–5813 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Yoo, S.J. et al Purification and characterization of the heat shock proteins HsIV and HsIU that form a new ATP-dependent protease in Escherichia coli. J. Biol.Chem. 271, 14035–14040 (1996).

    Article  CAS  PubMed  Google Scholar 

  10. Fleischmann, R.D. et al. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269, 496–512 (1995).

    Article  CAS  PubMed  Google Scholar 

  11. Highlander, S.K., Wickersham, E.A., Garza, O. & Weinstock, G.M. Expression of the Pasteurella haemolytica leukotoxin is inhibited by a locus that encodes an ATP-binding cassette homolog. Infect. Immun. 61, 3942–3951 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Lupas, A., Zwickl, P. & Baumeister, W. Proteasome sequences in eubacteria. Trends Biochem. Sci. 19, 533–534 (1994).

    Article  CAS  PubMed  Google Scholar 

  13. Slack, F.J., Serror, P., Joyce, E. & Sonenshein, A.L. A gene required for nutritional repression of the Bacillus subtilis dipeptide permease operon. Mol. Microbiol. 15, 689–702 (1995).

    Article  CAS  PubMed  Google Scholar 

  14. Becker, J. & Brendel, M. Molecular characterization of the xerC gene of Lactobacillus Leichmannii encoding a site-specific recombinase and two adjacent heat shock genes. Curr. Microbiol. 32, 232–236 (1996).

    Article  CAS  PubMed  Google Scholar 

  15. Schirmer, E.G., Glover, J.R., Singer, M.A. & Lindquist, S. HSP100/Clp proteins: a common mechanism explains diverse functions. Trends Biochem. Sci. 21, 289–296 (1996).

    Article  CAS  PubMed  Google Scholar 

  16. Chuang, S.E., Burland, V., Plunkett, G.d., Daniels, D.L. & Blattner, F.R. Sequence analysis of four new heat-shock genes constituting the hslTS/ibpAB and hslVU operons in Escherichia coll. Gene 134, 1–6 (1993).

    Article  CAS  PubMed  Google Scholar 

  17. Maurizi, M.R., Thompson, M.W., Singh, S.K. & Kirn, S.H., Clp protease from Escherichia coli. Meths. Enzymol. 244, 314–331 (1994).

    Article  CAS  Google Scholar 

  18. Dahlmann, B. et al. The multicatalytic proteinase (prosome) is ubiquitous from eukaryotes to archaebacteria. FEBS Letters 251, 125–131 (1989).

    Article  CAS  PubMed  Google Scholar 

  19. Tamura, T. et al. The first characterization of a eubacterial proteasome - the 20S complex of Rhodococcus. Curr. Biol. 5, 766–774 (1995).

    Article  CAS  PubMed  Google Scholar 

  20. Tanahashi, N., Tsurumi, C., Tamura, T. & Tanaka, K. Molecular structures of 20S and 26S proteasomes. Enzyme Prot. 47, 241–251 (1993).

    Article  CAS  Google Scholar 

  21. Lupas, A., Koster, A.J. & Baumeister, W. Structural features of 26S and 20S proteasomes. Enzyme Prot. 47, 252–273 (1993).

    Article  CAS  Google Scholar 

  22. Pühler, G. et al. Subunit stoichiometry and three-dimensional arrangement in proteasomes from Thermoplasma acidophilum. EMBO J. 11, 1607–1616 (1992).

    Article  PubMed  PubMed Central  Google Scholar 

  23. Waxman, L, Fagan, J.M. & Goldberg, A.L. Demonstration of two distinct high molecular weight proteases in rabbit reticulocytes, one of which degrades ubiquitin conjugates. Biol. Chem. 262, 2451–2457 (1987).

    CAS  Google Scholar 

  24. Hough, R., Pratt, G. & Rechsteiner, M. Purification of two high molecular weight proteases from rabbit reticulocyte lysate. J. Biol. Chem. 262, 8303–8313 (1987).

    CAS  PubMed  Google Scholar 

  25. Ganoth, D., Leshinsky, E., Eytan, E. & Hershko, A. A multicomponent system that degrades proteins conjugated to ubiquitin: resolution of factors and evidence for ATP-dependent complex formation. J. Biol. Chem. 263, 12412–12419 (1988).

    CAS  PubMed  Google Scholar 

  26. Hershko, A. & Ciechanover, A. The ubiquitin system for protein degradation. Ann. Rev. Biochem. 61, 761–807 (1992).

    Article  CAS  PubMed  Google Scholar 

  27. Peters, J.M., Cejka, Z., Harris, J.R., Kleinschmidt, J.A. & Baumeister, W. Structural features of the 26-S proteasome complex. J. Mol. Biol. 234, 932–937 (1993).

    Article  CAS  PubMed  Google Scholar 

  28. Coux, O., Tanaka, K. & Goldberg, A.L. Structure and functions of the 20S and 26S proteasomes. Ann. Rev. Biochem 65, 801–647 (1996).

    Article  CAS  PubMed  Google Scholar 

  29. Seemüller, E., Lupas, A. & Baumeister, W. Autocatalytic processing of the 20S proteasome. Nature 382, 468–470 (1996).

    Article  PubMed  Google Scholar 

  30. Kessel, M. et al. Homology in structural organization between E. coli CIpAP protease and the eukaryotic 26 S proteasome. J. Mol. Biol. 250, 587–594 (1995).

    Article  CAS  PubMed  Google Scholar 

  31. Löwe, J. et al. Crystal structure of the 20S proteasome from the archaeon I acidophilum at 3.4 A resolution. Science 268, 533–539 (1995).

    Article  PubMed  Google Scholar 

  32. Müller, S., Goldie, K.N., Bürki, R., Häring, R. & Engel, A. Factors influencing the precision of quantitative scanning transmission electron microscopy. Ultramicroscopy 46, 317 (1992).

    Article  Google Scholar 

  33. Engel, A. & Colliex, C. Application of scanning transmission electron microscopy to the study of biological structure. Curr. Opin. Biotechnol. 4, 403–411 (1993).

    Article  CAS  PubMed  Google Scholar 

  34. Gottesman, S. et al. Conservation of the regulatory subunit for the Clp ATP-dependent protease in prokaryotes and eukaryotes. Proc. Natl. Acad. Sci. USA 87, 3513–3517 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Zwickl, P., Kleinz, J. & Baumeister, W. Critical elements in proteasome assembly. Nature Struct. Biol. 1, 765–770 (1994).

    Article  CAS  PubMed  Google Scholar 

  36. Nederlof, P.M., Wang, H.R. & Baumeister, W. Nuclear localization signals of human and Thermoplasma cc subunits are functional in vitro. Proc. Natl. Acad. Sci. USA 92, 12060–12064 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Shin, D.H., Lee, C.S., Chung, C.H. & Suh, S.W. Molecular symmetry of the CIpP component of the ATP-dependent Clp Protease, an Escherichia coli homolog of the 20S proteasome. J. Mol. Biol. 262, 71–76 (1996).

    Article  CAS  PubMed  Google Scholar 

  38. Weissman, J.S., Sigler, P.B. & Horwich, A.L. From the cradle to the grave: ring complexes in the life of a protein. Science 268, 523–524 (1995).

    Article  CAS  PubMed  Google Scholar 

  39. Kelman, Z., Finkelstein, J. & O′Donnell, M. Protein structure. Why have six-fold symmetry? Curr. Biol. 5, 1239–1242 (1995).

    Article  CAS  PubMed  Google Scholar 

  40. Wawrzynow, A. et al. The CIpX heat-shock protein of Escherichia coli, the ATP-dependent substrate specificity component of the CIpP-CIpX protease, is a novel molecular chaperone. EMBOJ. 14, 1867–1877 (1995).

    Article  CAS  Google Scholar 

  41. Wickner, S. et al. A molecular chaperone, ClpA, functions like DnaK and DnaJ. Proc. Natl.Acad.Sci. USA 91, 12218–12222 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Missiakas, D., Schwager, F., Betton, J.-M., Georgopoulos, C. & Raina, S. Identification and characterization of HslV HslU (CIpQ CIpY) proteins involved in overall proteolysis of misfolded proteins in Escherichia coli. EMBO J., in press.

  43. Hegerl, R. & Altbauer, A. The “EM” program system. Ultramicroscopy 9, 109–116 (1982).

    Article  CAS  PubMed  Google Scholar 

  44. Saxton, W.O., Pitt, T.J. & Horner, M. Digital image processing: the Semper system. Ultramicroscopy 4, 343–354 (1979).

    Article  Google Scholar 

  45. Frank, J., Bretaudiere, J.P., Carazo, J.M., Verschoor, A. & Wagenknecht, T. Classification of images of biomolecular assemblies: a study of ribosomes and ribosomal subunits of Escherichia coli. J. Microscopy 150, 99–115 (1988).

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, 02115, USA

    Markus Rohrwild, H.-C. Huang & Alfred L. Goldberg

  2. Abteilung Molekulare Strukturbiologie, Max-Planck-lnstitut fur Biochemie, D-82152, Martinsried, Germany

    Günter Pfeifer, Ute Santarius & Wolfgang Baumeister

  3. M.E. Müller-Institute for Structural Biology, Biozentrum of the University, CH-4056, Basel, Switzerland

    Shirley A. Müller & Andreas Engel

Authors
  1. Markus Rohrwild
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Correspondence to Alfred L. Goldberg.

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Rohrwild, M., Pfeifer, G., Santarius, U. et al. The ATP-dependent HslVU protease from Escherichia coli is a four-ring structure resembling the proteasome. Nat Struct Mol Biol 4, 133–139 (1997). https://doi.org/10.1038/nsb0297-133

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