Conditional on perforin-dependent delivery to the nucleus of target cells, the cytolytic granule serine protease granzyme B (GrB) plays a central role in eliciting the nuclear events of apoptosis, as shown by the fact that reducing GrB nuclear entry prevents nuclear apoptosis. Apart from a requirement for cytosolic factors and lack of dependence on the guanine-nucleotide-binding protein Ran, little is known regarding the nuclear import pathway of GrB. In this study we use quantitative yeast two-hybrid and direct binding assays to show that GrB can be recognized independently by either of the nuclear import receptor family members importin (IMP) alpha and beta1, but that these proteins either alone or in combination cannot replace exogenous cytosol to reconstitute GrB nuclear import in vitro. Whereas antibodies to IMP(alpha) inhibit transport, indicating that IMP(alpha) is required for GrB nuclear import, those to IMP(beta) enhance transport, implying that IMP(beta) inhibits GrB nuclear import; consistent with this, the addition of recombinant IMP(beta) but not IMP(alpha) reduces maximal nuclear accumulation in the presence of cytosol. Intriguingly, complexation of GrB with its specific serpin inhibitor PI-9 was found to prevent recognition by IMP(beta) but not by IMP(alpha), and eliminate the apparent requirement for IMP(alpha) for nuclear import. We conclude that GrB nuclear import exhibits complex regulation by IMPs; that heterodimerization with PI-9 can modulate the interaction has implications for protection against apoptosis.
(c) 2005 Wiley-Liss, Inc.