Ubiquitin-mediated proteolysis centers in HeLa cells: indication from studies of an inhibitor of the chymotrypsin-like activity of the proteasome

Abstract

HeLa cells growing in vitro were treated with the peptidyl aldehyde inhibitor of the chymotrypsin-like activity of the proteasome N-benzyloxycarbonyl-Ile-Glu(O-t-butyl)-Ala-leucinal (PSI). Immunofluorescence studies of treated cells revealed the formation of massive perinuclear aggregates rich in ubiquitin and proteasomal antigens, which on the ultrastructural level appeared as perinuclear aggregates of electron-dense material, usually in the vicinity of Golgi cisternae. Histochemical studies disclosed that these cells contained protein-rich perinuclear aggregates detected by amido black staining, while unusual accumulations of lipids, carbohydrates, or nucleic acids were not present. Inhibition of protein synthesis by cycloheximide prevented the formation of aggregates, whereas microtubule disruption by nocodazole induced a dispersion of the aggregates. We hypothesize that aggregates induced by PSI treatment correspond to accumulations of proteasome-substrate complexes in a well-defined region, where the proteolytic processes of the ubiquitin-proteasome pathway seem to be somehow centered. We propose to call this region the proteolysis center.