Subunit Topology of Two 20S Proteasomes from Haloferax volcanii (original) (raw)

Haloferax volcanii, a halophilic archaeon, synthesizes three different proteins (␣1, ␣2, and ␤) which are classified in the 20S proteasome superfamily. The ␣1 and ␤ 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 ␤ in a complex with an overall structure and peptide-hydrolyzing activity comparable to those of the previously described ␣1-␤ proteasome. Supplementing buffers with 10 mM CaCl 2 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-␤ and ␣1-␣2-␤ proteasomes, were identified during stationary phase. Cross-linking of these enzymes, coupled with available structural information, suggested that the ␣1-␤ proteasome was a symmetrical cylinder with ␣1 rings on each end. In contrast, the ␣1-␣2-␤ 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.