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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

Lara S. Madding ,^, Joshua K. Michel, 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 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 +ß1+ß2 CP assembled at 105°C was more thermostable than either the +ß1+ß2 version assembled at 90°C or the +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 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.

Published ahead of print on 17 November 2006.

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

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.