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 PY _NLS (proline-tyrosine NLS) motifs—previously described only in animals and fungi—remains unexplored. Here, we present the first systematic characterization of PY _NLS motifs in plants, demonstrating their functionality and divergence from known eukaryotic models. Using transient expression in tobacco and stable Arabidopsis lines, we show that PY _NLS motifs from the jasmonate signaling protein JAZ1 and a previously uncharacterized PLA₂-like protein mediate Karyopherin β2 (Kapβ2)-dependent nuclear import. Through site-directed mutagenesis, structural modeling, and interaction assays, we define key sequence and structural determinants of functional PY _NLS activity, including an extended linker between binding epitopes and a short α-helix upstream of the conserved PY dipeptide. These features distinguish plant PY _NLS motifs from their counterparts in animals and fungi. Leveraging these criteria, we identified 125 Arabidopsis proteins with predicted functional PY _NLSs , encompassing transcription factors, RNA-binding proteins, and components involved in calcium signaling. Notably, Kapβ2 interaction also modulated condensate formation by cytoplasmic JAZ1, indicating a dual role for Kapβ2 in nuclear import and phase separation. Our findings reveal a plant-specific PY _NLS architecture and highlight Kapβ2 as a multifunctional regulator of nuclear organization and biomolecular condensates, bridging a key gap in our understanding of plant nucleocytoplasmic transport.