Nuclear LC3 Associates with Slowly Diffusing Complexes that Survey the Nucleolus

MAP1LC3B (microtubule‐associated protein 1 light chain 3, LC3) is a key component of the autophagy pathway, contributing to both cargo selection and autophagosome formation in the cytoplasm. Emerging evidence suggests that nuclear forms of LC3 are also functionally important; however, the mechanisms that facilitate the nuclear targeting and trafficking of LC3 between the nucleus and cytoplasm under steady‐state conditions are poorly understood. In this study, we examine how residues known to regulate the interactions between LC3 and other proteins or RNA (F52 L53, R68‐R70 and G120) contribute to its nuclear targeting, nucleocytoplasmic transport and association with nucleoli and other nuclear components. We find that residues F52 L53 and R68‐70, but not G120, regulate targeting of LC3 to the nucleus, its rates of nucleocytoplasmic transport and the apparent sizes of LC3‐associated complexes in the nucleus inferred from fluorescence recovery after photobleaching (FRAP) measurements. We also show that LC3 is enriched in nucleoli and its triple arginine motif is especially important for nucleolar targeting. Finally, we identify a series of candidate nuclear LC3‐interacting proteins using mass spectrometry, including MAP1B, tubulin and several 40S ribosomal proteins. These findings suggest LC3 is retained in the nucleus in association with high‐molecular weight complexes that continuously scan the nucleolus. A key component of the autophagy pathway, microtubule‐associated protein 1 light chain 3 (LC3), contributes to both autophagosome formation and cargo selection. LC3 is also present in the nucleus under basal conditions, but the mechanisms that facilitate the nuclear targeting and trafficking of LC3 between the nucleus and cytoplasm are poorly understood. Here, we show that LC3 is retained in the nucleus in association with high‐molecular weight complexes, and also accesses the nucleolus. In addition, we identify a series of candidate nuclear LC3‐interacting proteins.