Role of the β1 Subunit in the Function and Stability of the 20S Proteasome in the Hyperthermophilic ArchaeonPyrococcus furiosus

Abstract

The hyperthermophilic archaeon Pyrococcus furiosus genome encodes three proteasome component proteins: one ␣ protein (PF1571) and two ␤ proteins (␤1-PF1404 and ␤2-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 ␤1 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 ␤1 protein relative to ␤2 into the 20S proteasome (core particle [CP]) increased with increasing temperature for both native and recombinant versions. For the recombinant enzyme, the ␤2/␤1 ratio varied linearly with temperature from 3.8, when assembled at 80°C, to 0.9 at 105°C. The recombinant ␣؉␤1؉␤2 CP assembled at 105°C was more thermostable than either the ␣؉␤1؉␤2 version assembled at 90°C or the ␣؉␤2 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 ␤1 proteins neither yielded a large proteasome complex nor demonstrated any significant activity. These results indicate that the ␤1 subunit in the P. furiosus 20S proteasome plays a thermostabilizing role and influences biocatalytic properties, suggesting that ␤ subunit composition is a factor in archaeal proteasome function during thermal stress, when polypeptide turnover is essential to cell survival.

The proteasomal system

“…HsC2/HsPROS30 30.2 Kania et al (1996Kania et al ( , 2005 a7 C1/ Prs1 HsC8 28.4 Bose et al (2004), Gerards et al (1997), Gerards et al (1998) and Shu et al (2003) b1 Pre3 HsDelta/Y 25.3 (21.9) Chung et al (2000), Lequeu et al (2005), Madding et al (2007) b1i -Lmp2 23.2 (20.9) Dissemond et al (2003), , and Krause et al (2006) b2 Pup1 Z 30.0 (24.5) Schweisguth (1999), Ramos et al (2004) and Baldisserotto et al (2007) b2i -Mecl1 28.9 (23.8) Scheffler et al (2008) and Eleuteri et al (1997) b3 Pup3…”

Proteasomes and protein conjugation across domains of life

“…Pyrococcus furiosus also encodes three CP subunits (α, β1 and β2). Of these, β1 subunit transcript levels are upregulated during heat shock, and CPs with the greatest ratio of β1/β2 are the most thermostable 113 . Thus, P. furiosus might incorporate β1 into CPs to enhance proteasome function during thermal stress.…”

Polypeptide and Polysaccharide Processing in Hyperthermophilic Microorganisms

“… These results indicate that the β1 protein in the P. furiosus 20S proteasome plays a thermostabilizing role at supraoptimal temperatures and suggests that subunit composition can be a factor in proteasome function during thermal stress when polypeptide turnover is essential to cell survival (Madding et al, 2007).…”

“…(5) Role of 20S proteasome β protein in the hyperthermophilic archaeon Pyrococcus furiosus during thermal stress  Transcriptional analysis of P. furiosus subjected to heat shock (shift from 90 to 105°C) showed that expression of the β2 gene (PF0159) in the archaeal proteasome was affected differently than the β1 gene (PF1404), suggesting different roles in thermal stress response for the subunits (Madding et al, 2007).…”