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Journal of Bacteriology, January 2007, p. 583-590, Vol. 189, No. 2
0021-9193/07/$08.00+0     doi:10.1128/JB.01382-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Role of the ß1 Subunit in the Function and Stability of the 20S Proteasome in the Hyperthermophilic Archaeon Pyrococcus furiosus{triangledown}

Lara S. Madding ,{dagger},{ddagger} Joshua K. Michel,{dagger} Keith R. Shockley,§ Shannon B. Conners, Kevin L. Epting, Matthew R. Johnson,|| and Robert M. Kelly*

Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina 27695-7905

Received 30 August 2006/ Accepted 27 October 2006

The hyperthermophilic archaeon Pyrococcus furiosus genome encodes three proteasome component proteins: one {alpha} protein (PF1571) and two ß proteins (ß1-PF1404 and ß2-PF0159), as well as an ATPase (PF0115), referred to as proteasome-activating nucleotidase. Transcriptional analysis of the P. furiosus dynamic heat shock response (shift from 90 to 105°C) showed that the ß1 gene was up-regulated over twofold within 5 minutes, suggesting a specific role during thermal stress. Consistent with transcriptional data, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that incorporation of the ß1 protein relative to ß2 into the 20S proteasome (core particle [CP]) increased with increasing temperature for both native and recombinant versions. For the recombinant enzyme, the ß2/ß1 ratio varied linearly with temperature from 3.8, when assembled at 80°C, to 0.9 at 105°C. The recombinant {alpha}+ß1+ß2 CP assembled at 105°C was more thermostable than either the {alpha}+ß1+ß2 version assembled at 90°C or the {alpha}+ß2 version assembled at either 90°C or 105°C, based on melting temperature and the biocatalytic inactivation rate at 115°C. The recombinant CP assembled at 105°C was also found to have different catalytic rates and specificity for peptide hydrolysis, compared to the 90°C assembly (measured at 95°C). Combination of the {alpha} and ß1 proteins neither yielded a large proteasome complex nor demonstrated any significant activity. These results indicate that the ß1 subunit in the P. furiosus 20S proteasome plays a thermostabilizing role and influences biocatalytic properties, suggesting that ß subunit composition is a factor in archaeal proteasome function during thermal stress, when polypeptide turnover is essential to cell survival.

* Corresponding author. Mailing address: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695-7905. Phone: (919) 515-6396. Fax: (919) 515-3465. E-mail: rmkelly{at}eos.ncsu.edu.

{triangledown} Published ahead of print on 17 November 2006.

{dagger} L.S.M. and J.K.M. contributed equally to this work.

{ddagger} Present address: Dept. of Pathology, Duke Univ. Medical Center, Durham, NC 27710.

§ Present address: The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609.

Present address: SAS Corporation, Cary, N.C.

|| Present address: Wyeth Pharmaceuticals, 4300 Oak Park, Sanford, NC 27330.

Journal of Bacteriology, January 2007, p. 583-590, Vol. 189, No. 2
0021-9193/07/$08.00+0     doi:10.1128/JB.01382-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Humbard, M. A., Zhou, G., Maupin-Furlow, J. A. (2009). The N-Terminal Penultimate Residue of 20S Proteasome {alpha}1 Influences its N{alpha} Acetylation and Protein Levels as Well as Growth Rate and Stress Responses of Haloferax volcanii. J. Bacteriol. 191: 3794-3803 [Abstract] [Full Text]  


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