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Journal of Bacteriology, September 1999, p. 5814-5824, Vol. 181, No. 18
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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

Heather L. Wilson, Henry C. Aldrich, and Julie Maupin-Furlow*

Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611-0700

Received 6 May 1999/Accepted 15 July 1999

A 20S proteasome, composed of alpha 1 and beta  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 alpha 1beta -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 alpha 1beta -proteasome also hydrolyzed insulin B-chain protein. Removal of NaCl inactivated the CL activity of the alpha 1beta -proteasome and dissociated the complex into monomers. Rapid equilibration of the monomers into buffer containing 2 M NaCl facilitated their reassociation into fully active alpha 1beta -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 beta -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 (Ki = 0.2 and 8 µM, respectively). In addition to the genes encoding the alpha 1 and beta  subunits, a gene encoding a second alpha -type proteasome protein (alpha 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 alpha 1beta -proteasome resulted in the copurification of the alpha 2 protein with the alpha 1 and beta  subunits in nonstoichometric ratios as cylindrical particles of four stacked rings of 600 kDa with CL activity rates similar to the alpha 1beta -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 alpha  subunits in this halophilic archaeon.

* Corresponding author. Mailing address: Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611-0700. Phone: (352) 392-4095. Fax: (352) 392-5922. E-mail: jmaupin{at}micro.ifas.ufl.edu.

Journal of Bacteriology, September 1999, p. 5814-5824, Vol. 181, No. 18
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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