Adriamycin (ADM), an
anthracycline anticancer agent, is selectively stored in the nuclei of a variety of proliferating cells, but the precise mechanism of specific nuclear transport of ADM is not well known. Recently, we demonstrated that ADM shows high binding affinity to the cytoplasmic proteasomes of L1210 mouse
leukemia cells and that taken up ADM by the cells selectively binds to proteasomes. Nuclear targeting of
proteasome in proliferating cells may be mediated by the
nuclear localization signals that are found in several of the alpha-type subunits of the
20S proteasome. To confirm nuclear transport of the ADM-
proteasome complex, we synthesized a photoactive ADM analogue, N-(p-azidohenzoyl)-ADM, and generated a photoaffinity-labeled
proteasome complex. The
26S proteasome purified from the cytosol of L1210 cells had a high affinity to N-(p-azidobenzoyl)-ADM. SDS-PAGE analysis of the photoaffinity-labeled
proteasome showed that low molecular weight bands (approximately 21-31 kDa) of
20S proteasome had the highest photoaffinity. The photoaffinity-labeled
proteasome was distributed in the cytoplasm and nuclei of
digitonin-permeabilized L1210 and B-16 mouse
melanoma cells in the presence of the cytosolic fraction and
ATP. The rate of nuclear translocation of the
proteasome was low in the absence of
ATP. These results suggest that the
proteasome is a specific translocator of ADM from the cytoplasm to the nucleus and that
20S proteasome components are the dominant ADM-binding sites. The nuclear transport of ADM-
proteasome complex is regulated by an
ATP-dependent nuclear pore-mediated mechanism.