The 26S proteasome holoenzyme is formed by the association of a 20S core particle (CP) with a 19S regulatory particle (RP). The CP-RP interaction is labile and subject to regulation in vivo, but the factors controlling this association are poorly understood. Here we describe an in vitro proteasome reconstitution assay and a high-resolution, two-dimensional gel electrophoresis system. Using these techniques, we find that a yeast CP-RP complex can contain a substoichiometric amount of tightly bound, essentially non-exchangeable ATP. However, this nucleotide is dispensable for gating of the CP channel, provided that the CP-RP complex is preserved by the Ecm29 protein. Unexpectedly, proteasome inhibitors are potent in stabilizing proteasomes against the dissociation of CP-RP. These data indicate that active sites of the CP communicate with bound RP, despite their spatial separation. We propose that ongoing protein degradation may suppress proteasome disassembly, thereby enhancing the processivity of proteolysis.