The carboxyl terminus of interferon-gamma contains a functional polybasic nuclear localization sequence

Area of Science:

• Molecular Biology
• Cell Biology
• Immunology

Background:

• Interferon-gamma (IFN-gamma) signaling involves the JAK/STAT pathway, but its nuclear translocation mechanism and the role of ligand nuclear import remain unclear.
• Understanding IFN-gamma's nuclear import is crucial for elucidating its full spectrum of biological activities and intracellular functions.

Purpose of the Study:

• To identify and characterize the nuclear localization signal (NLS) of interferon-gamma (IFN-gamma).
• To investigate the mechanism and pathway involved in the nuclear import of IFN-gamma.

Main Methods:

• Nuclear import assays using digitonin-permeabilized cells with allophycocyanin (APC) fused to IFN-gamma peptides.
• Competition assays using specific IFN-gamma peptides and SV40 large T-antigen NLS peptide.
• Energy-dependence studies using ATP and GTP, and deletion analysis of the IFN-gamma sequence.

Main Results:

• A polybasic sequence (126RKRKRSR132) in the COOH terminus of IFN-gamma was identified as necessary and sufficient for nuclear localization.
• Nuclear import of IFN-gamma and its NLS-containing peptide was energy-dependent (ATP and GTP required) and competed by SV40 T-NLS peptide.
• Intact IFN-gamma coupled to APC also demonstrated nuclear import, inhibited by the IFN-gamma NLS peptide and SV40 T-NLS peptide, suggesting Ran/importin pathway involvement.

Conclusions:

• The COOH-terminal sequence 126RKRKRSR132 functions as a nuclear localization sequence (NLS) for IFN-gamma.
• IFN-gamma nuclear import utilizes the energy-dependent Ran/importin pathway, similar to other NLS-containing proteins.
• The nuclear import of IFN-gamma suggests a direct intracellular role, potentially involving interactions within the nucleus mediated by its NLS.