FUNCTIONAL-ANALYSIS OF EUKARYOTIC 20S PROTEASOME NUCLEAR-LOCALIZATIONSIGNAL

The 20S proteasome is widely viewed at as a cytoplasmic multicatalytic proteinase complex: immunocytochemical investigations, however, show that proteasomes are localized in the cytoplasm as well as in the nucl eus within the same cell. Strong nuclear accumulation of proteasomes i s observed in rapidly dividing cells such as in the early stages of Dr osophila embryogenesis and in tumorigenic cells. In fact, dependent on the metabolic state of a certain tissue or cell type its cellular dis tribution appears differentially regulated. Several of the proteasomal alpha-type subunits carry putative nuclear localization signals which may or may not take part in the regulation of the intracellular distr ibution of 20S proteasomes. We have examined the functional role of th e putative nuclear localization signal (NLS) -KKKQKK-in the Drosophila PROS-28.1 subunit by deletion mutagenesis and transfection experiment s, Linkage of the putative PROS-28.1 NLS to BSA as reporter protein an d in vibro import studies with permeabilized mouse NIH 3T3 cells show that this NLS is able to induce complete translocation of the reporter protein into the cell nucleus. For analysis of the NLS within the 28- kDa subunit, cDNA deletion constructs were cloned into a pSG5 expressi on vector and transiently transfected into mouse fibroblast cells. Whe reas the deletion of the NLS alone resulted only in a slight impairmen t of subunit transport into the nucleus, removal of the C-terminal 96 amino acid residues abolished nuclear translocation completely. (C) 19 96 Academic Press, Inc.