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Subunit Topology of Two 20S Proteasomes from Haloferax volcanii

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 (1, 2, and ß) which are classified in the 20S proteasome superfamily. The 1 and ß proteins alone form active 20S proteasomes; the role of 2, however, is not clear. To address this, 2 was synthesized with an epitope tag and purified by affinity chromatography from recombinant H. volcanii. The 2 protein copurified with 1 and ß in a complex with an overall structure and peptide-hydrolyzing activity comparable to those of the previously described 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 1-ß and 1-2-ß proteasomes, were identified during stationary phase. Cross-linking of these enzymes, coupled with available structural information, suggested that the 1-ß proteasome was a symmetrical cylinder with 1 rings on each end. In contrast, the 1-2-ß proteasome appeared to be asymmetrical with homo-oligomeric 1 and 2 rings positioned on separate ends. Inter--subunit contacts were only detected when the ratio of 1 to 2 was perturbed in the cell using recombinant technology. These results support a model that the ratio of proteins may modulate the composition and subunit topology of 20S proteasomes in the cell.

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