In vivo characterization of the proteasome regulator PA28

A proteasome regulator, termed PA28, has been shown to modulate peptidase activities of the proteasomes in vitro. Two different but homologous PA28 molecules, designated as PA28alpha and PA28beta, have been cloned. Both alpha and beta polypeptides of PA28 are found in PA28 complexes isolated from cells, indicating that both are constituents of functional PA28 complexes. Using antisera specific to PA28alpha, PA28beta, and epitope-tagged PA28 molecules, we show that expression of PA28alpha and PA28beta is coordinately induced by various cytokines in different cell lines and that PA28 subunits and proteasomes have almost identical half-lives. In addition, we show that PA28 complexes are associated with 20 S but not 26 S proteasomes in vivo. Moreover, we demonstrate that PA28 complex is a heterohexamer composed of both alpha and beta subunits with a stoichiometry of alpha3beta3 in an alternating order.