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Journal of Bacteriology, January 2003, p. 165-174, Vol. 185, No. 1
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.1.165-174.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Subunit Topology of Two 20S Proteasomes from Haloferax volcanii

Steven J. Kaczowka and Julie A. Maupin-Furlow*

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

Received 3 July 2002/ Accepted 2 October 2002

Haloferax volcanii, a halophilic archaeon, synthesizes three different proteins ({alpha}1, {alpha}2, and ß) which are classified in the 20S proteasome superfamily. The {alpha}1 and ß proteins alone form active 20S proteasomes; the role of {alpha}2, however, is not clear. To address this, {alpha}2 was synthesized with an epitope tag and purified by affinity chromatography from recombinant H. volcanii. The {alpha}2 protein copurified with {alpha}1 and ß in a complex with an overall structure and peptide-hydrolyzing activity comparable to those of the previously described {alpha}1-ß proteasome. Supplementing buffers with 10 mM CaCl2 stabilized the halophilic proteasomes in the absence of salt and enabled them to be separated by native gel electrophoresis. This facilitated the discovery that wild-type H. volcanii synthesizes more than one type of 20S proteasome. Two 20S proteasomes, the {alpha}1-ß and {alpha}1-{alpha}2-ß proteasomes, were identified during stationary phase. Cross-linking of these enzymes, coupled with available structural information, suggested that the {alpha}1-ß proteasome was a symmetrical cylinder with {alpha}1 rings on each end. In contrast, the {alpha}1-{alpha}2-ß proteasome appeared to be asymmetrical with homo-oligomeric {alpha}1 and {alpha}2 rings positioned on separate ends. Inter-{alpha}-subunit contacts were only detected when the ratio of {alpha}1 to {alpha}2 was perturbed in the cell using recombinant technology. These results support a model that the ratio of {alpha} proteins may modulate the composition and subunit topology of 20S proteasomes in the cell.

* 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}ufl.edu.

Journal of Bacteriology, January 2003, p. 165-174, Vol. 185, No. 1
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.1.165-174.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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