NUCLEAR TRANSPORT OF GRANZYME-B (FRAGMENTIN-2) - DEPENDENCE ON PERFORIN IN-VIVO AND CYTOSOLIC FACTORS IN-VITRO

Cytotoxic T and natural killer cells are able to kill their target cel ls through synergistic action of the pore-forming protein perforin and the serine protease granzyme B, resulting in very distinctive nuclear changes typical of apoptosis. Whereas perforin acts at the membrane, granzyme B appears to be both capable of entering the cytoplasm of tar get cells and accumulating in isolated nuclei, In this study we examin e nuclear transport of fluoresceinated granzyme B both in vivo in inta ct cells in the presence of perforin and in vitro in semipermeabilized cells using confocal laser scanning microscopy. Granzyme B alone was observed to enter the cytoplasm of intact cells but did not accumulate in nuclei, In the presence of sublytic concentrations of perforin, ho wever, it accumulated strongly in intact cell nuclei to levels maximal ly about 1.5 times those in the cytoplasm after about 2.5 h, In vitro nuclear transport assays showed maximal levels of nuclear and nucleola r accumulation of granzyme B of about 2.5- and 3-fold those in the cyt oplasm, In contrast to signal-dependent nuclear accumulation of SV40 l arge tumor antigen (T-Ag) fusion proteins in vitro, nuclear/nucleolar import of granzyme B was independent of ATP and not inhibitable by the non-hydrolyzable GTP analog GTP gamma S (guanosine 5'-O-(3-thiotripho sphate)). Similar to T-Ag fusion proteins, however, granzyme B nuclear and nucleolar accumulation was dependent on exogenously added cytosol , Specific inhibitors of granzyme B protease activity had no effect on nuclear/nucleolar accumulation, implying that proteolytic activity wa s not essential for nuclear targeting, The results imply that granzyme B (32 kDa) may be transported from the cytoplasm to the nucleus throu gh passive diffusion and accumulate by binding to nuclear/nucleolar fa ctors in a cytosolic factor-mediated process, Active and passive nucle ar transport properties were normal in the presence of unlabeled granz yme B, implying that the nuclear envelope and pore complex are not gra nzyme B substrates.