title: Role of the beta1 subunit in the function and stability of the 20S proteasome in the hyperthermophilic archaeon Pyrococcus furiosus.
abstract: The hyperthermophilic archaeon Pyrococcus furiosus genome encodes three proteasome component proteins: one alpha protein (PF1571) and two beta proteins (beta1-PF1404 and beta2-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 degrees C) showed that the beta1 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 beta1 protein relative to beta2 into the 20S proteasome (core particle [CP]) increased with increasing temperature for both native and recombinant versions. For the recombinant enzyme, the beta2/beta1 ratio varied linearly with temperature from 3.8, when assembled at 80 degrees C, to 0.9 at 105 degrees C. The recombinant alpha+beta1+beta2 CP assembled at 105 degrees C was more thermostable than either the alpha+beta1+beta2 version assembled at 90 degrees C or the alpha+beta2 version assembled at either 90 degrees C or 105 degrees C, based on melting temperature and the biocatalytic inactivation rate at 115 degrees C. The recombinant CP assembled at 105 degrees C was also found to have different catalytic rates and specificity for peptide hydrolysis, compared to the 90 degrees C assembly (measured at 95 degrees C). Combination of the alpha and beta1 proteins neither yielded a large proteasome complex nor demonstrated any significant activity. These results indicate that the beta1 subunit in the P. furiosus 20S proteasome plays a thermostabilizing role and influences biocatalytic properties, suggesting that beta subunit composition is a factor in archaeal proteasome function during thermal stress, when polypeptide turnover is essential to cell survival.
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695-7905, USA.
authors.forename: Lara S
authors.initials: LS
authors.lastname: Madding
authors.forename: Joshua K
authors.initials: JK
authors.lastname: Michel
authors.forename: Keith R
authors.initials: KR
authors.lastname: Shockley
authors.forename: Shannon B
authors.initials: SB
authors.lastname: Conners
authors.forename: Kevin L
authors.initials: KL
authors.lastname: Epting
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Johnson
authors.forename: Robert M
authors.initials: RM
authors.lastname: Kelly
mesh_terms: D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D002152:Calorimetry, Differential Scanning; D020745:Genome, Archaeal; D006358:Hot Temperature; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D020027:Pyrococcus furiosus / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D014158:Transcription, Genetic
chemical_list: D019843:Archaeal Proteins; D018832:Molecular Chaperones; D021122:Protein Subunits; D046988:Proteasome Endopeptidase Complex