Differential properties of two putative nuclear localization sequences found in the carboxyl-terminus of human...

Interferon-gamma (IFN-gamma), a protein that uses the Jak-Stat pathway for signal transduction, translocates rapidly to the nucleus in cells treated extracellularly with the cytokine. A nuclear localization sequence (NLS) has been identified and characterized in the C-terminus of IFN-gamma. Both human and murine IFN-gamma contain this NLS. We show in this report that human IFN-gamma (HuIFN-gamma) contains a second NLS at an upstream site, as determined in standard import assays using digitonin-permeabilized HeLa cells. The primary sequence, analogous with the NLS sequence identified in murine IFN-gamma, representing amino acids 122-132 of HuIFN-gamma was capable of mediating the nuclear import of the autofluorescent protein allophycocyanin (APC) in an energy-dependent manner. The second sequence, representing amino acids 78-92 of HuIFN-gamma, was also capable of mediating the nuclear import of APC in an energy-dependent manner but to a greatly reduced extent. The nuclear import of both sequences conjugated to APC was strongly blocked by competition with unconjugated HuIFN-gamma(122-132). Competition by the sequence HuIFN-gamma(78-92) effectively blocked the import of APC-conjugated HuIFN-gamma(78-92) but, at the same concentration, was not capable of inhibiting the nuclear import of APC-conjugated HuIFN-gamma(122-132), suggesting that HuIFN-gamma(78-92) was a less efficient NLS than HuIFN-gamma(122-132). This is consistent with >90% loss of antiviral activity of HuIFN-gamma lacking the downstream NLS in 122-132. The nuclear import of APC-conjugated HuIFN-gamma(122-132) was inhibited by a peptide containing the prototypical polybasic NLS of the SV40 T NLS, which suggests that the same Ran/importin cellular machinery is used in both cases.