Purification procedures determine the proteasome activation properties of REGγ (PA28γ)

The proteasome activation properties of recombinant REGgamma molecules depend on purification procedures. Prior to ammonium sulfate precipitation recombinant REGgamma activates the trypsin-like catalytic subunit of the proteasome; afterwards it activates all three catalytic subunits. The expanded activation specificity is accompanied by reduced stability of the REGgamma heptamer providing support for the idea that a "tight" REGgamma heptamer suppresses the proteasome's chymotrypsin-like and postglutamyl-preferring active sites. In an attempt to determine whether REGgamma synthesized in mammalian cells also exhibits restricted activation properties, extracts were prepared from several mammalian organs and cell lines. Surprisingly, endogenous REGgamma was found to be largely monomeric. In an alternate approach, COS7 cells were cotransfected with plasmids expressing FLAG-REGgamma and REGgamma. The expressed FLAG-REGgamma molecules were shown to form oligomers with untagged REGgamma subunits, and the mixed oligomers preferentially activated the proteasome's trypsin-like subunit. Thus, REGgamma molecules synthesized in mammalian cells also exhibit restricted activation properties. (C) 2004 Elsevier Inc. All rights reserved.