Role of the beta1 subunit in the function and stability of the 20S proteasome in the hyperthermophilic archaeon Pyrococcus furiosus.

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.