Halophilic 20S Proteasomes of the Archaeon Haloferax volcanii : Purification, Characterization, and Gene Sequence Analysis

Abstract

ABSTRACT A 20S proteasome, composed of α 1 and β subunits arranged in a barrel-shaped structure of four stacked rings, was purified from a halophilic archaeon Haloferax volcanii . The predominant peptide-hydrolyzing activity of the 600-kDa α 1 β-proteasome on synthetic substrates was cleavage carboxyl to hydrophobic residues (chymotrypsin-like [CL] activity) and was optimal at 2 M NaCl, pH 7.7 to 9.5, and 75°C. The α 1 β-proteasome also hydrolyzed insulin B-chain protein. Removal of NaCl inactivated the CL activity of the α 1 β-proteasome and dissociated the complex into monomers. Rapid equilibration of the monomers into buffer containing 2 M NaCl facilitated their reassociation into fully active α 1 β-proteasomes of 600 kDa. However, long-term incubation of the halophilic proteasome in the absence of salt resulted in hydrolysis and irreversible inactivation of the enzyme. Thus, the isolated proteasome has unusual salt requirements which distinguish it from any proteasome which has been described. Comparison of the β-subunit protein sequence with the sequence deduced from the gene revealed that a 49-residue propeptide is removed to expose a highly conserved N-terminal threonine which is proposed to serve as the catalytic nucleophile and primary proton acceptor during peptide bond hydrolysis. Consistent with this mechanism, the known proteasome inhibitors carbobenzoxyl-leucinyl-leucinyl-leucinal-H (MG132) and N -acetyl-leucinyl-leucinyl-norleucinal (calpain inhibitor I) were found to inhibit the CL activity of the H. volcanii proteasome ( K i = 0.2 and 8 μM, respectively). In addition to the genes encoding the α 1 and β subunits, a gene encoding a second α-type proteasome protein (α 2 ) was identified. All three genes coding for the proteasome subunits were mapped in the chromosome and found to be unlinked. Modification of the methods used to purify the α 1 β-proteasome resulted in the copurification of the α 2 protein with the α 1 and β subunits in nonstoichometric ratios as cylindrical particles of four stacked rings of 600 kDa with CL activity rates similar to the α 1 β-proteasome, suggesting that at least two separate 20S proteasomes are synthesized. This study is the first description of a prokaryote which produces two separate 20S proteasomes and suggests that there may be distinct physiological roles for the two different α subunits in this halophilic archaeon.