<i>Drosophila</i> Nup358 functions as a SUMO E3 ligase to promote the SUMOylation and nuclear import of the juvenile hormone receptor Methoprene‐tolerant

Juvenile hormone (JH) governs insect development and reproduction primarily through its intracellular receptor Methoprene-tolerant (Met). The nuclear translocation of Met is a critical step in activating the JH signaling pathway, yet the regulatory mechanism underlying this process remains incompletely understood. Here, we demonstrate that SUMOylation is essential for the JH-induced nuclear import of Met in Drosophila. Knockdown of the SUMO-conjugating enzyme Ubc9 or the nucleoporin Nup358, but not the canonical E3 ligase Su(var)2-10, blocked Met nuclear accumulation and suppressed JH signaling. We further identified three specific lysine residues (K298, K429, K526) within Met as the major SUMOylation sites. A SUMO-deficient Met mutant (3KR) was retained in the cytoplasm and exhibited impaired transcriptional activity and coactivator recruitment. Crucially, we identified Nup358 as the specific SUMO E3 ligase responsible for Met SUMOylation. Its E3 activity depends on SUMO-interacting motifs (SIMs) within a defined domain, which binds to Met. Our findings establish SUMOylation as a critical regulatory layer for Met nucleocytoplasmic trafficking and reveal a non-canonical role for Nup358 as a SUMO E3 ligase in Drosophila, suggesting a mechanism whereby Nup358 couples substrate SUMOylation with nuclear import during insect JH signaling.