Non-classical Nuclear Signal PY-NLS Mediates Karyopherinβ2-Dependent Nuclear Import and Condensate Formation in Plants

Nuclear import is a fundamental cellular process regulated by nuclear localization signals (NLSs) and their cognate transport receptors. While classical NLSs have been extensively characterized in plants, the functionality of non-classical PYNLS (proline-tyrosine NLS) motifs--previously described only in animals and fungi--remains unexplored. Here, we present the first systematic characterization of PYNLS motifs in plants, demonstrating their functionality and divergence from known eukaryotic models. Using transient expression in tobacco and stable Arabidopsis lines, we show that PYNLS motifs from the jasmonate signaling protein JAZ1 and a previously uncharacterized PLA2-like protein mediate Karyopherin {beta}2 (Kap{beta}2)-dependent nuclear import. Through site-directed mutagenesis, structural modeling, and interaction assays, we define key sequence and structural determinants of functional PYNLS activity, including an extended linker between binding epitopes and a short -helix upstream of the conserved PY dipeptide. These features distinguish plant PYNLS motifs from their counterparts in animals and fungi. Leveraging these criteria, we identified 125 Arabidopsis proteins with predicted functional PYNLSs, encompassing transcription factors, RNA-binding proteins, and components involved in calcium signaling. Notably, Kap{beta}2 interaction also modulated condensate formation by cytoplasmic JAZ1, indicating a dual role for Kap{beta}2 in nuclear import and phase separation. Our findings reveal a plant-specific PYNLS architecture and highlight Kap{beta}2 as a multifunctional regulator of nuclear organization and biomolecular condensates, bridging a key gap in our understanding of plant nucleocytoplasmic transport.