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Nuclear LC3 Associates with Slowly Diffusing Complexes that Survey the Nucleolus.

ABSTRACT: 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.

SUBMITTER: Kraft LJ

PROVIDER: S-EPMC4975375 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Nuclear LC3 Associates with Slowly Diffusing Complexes that Survey the Nucleolus.

Kraft Lewis J LJ Manral Pallavi P Dowler Jacob J Kenworthy Anne K AK

Traffic (Copenhagen, Denmark) 20160218 4

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 reg ...[more]

PMID: 26728248

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Project description:MOAP1 (modulator of apoptosis 1) is a BAX-binding protein tightly regulated by the ubiquitin-proteasome system. Apoptotic stimuli stabilize MOAP1 protein and facilitate its interaction with BAX to promote apoptosis. Here we show that in contrast to being resistant to apoptotic stimuli, MOAP1-deficient cells are hypersensitive to cell death mediated by starvation rendered by EBSS treatment. MOAP1-deficient cells exhibited impairment in macroautophagy/autophagy signaling induced by EBSS. Mechanistic analysis revealed that MOAP1-deficient cells had no notable defect in the recruitment of the pre-autophagosomal phosphatidylinositol-3-phosphate (PtdIns3P)-binding proteins, ZFYVE1/DFCP1 and WIPI2, nor in the LC3 lipidation mechanism regulated by the ATG12-ATG5-ATG16L1 complex upon EBSS treatment. Interestingly, MOAP1 is required for facilitating efficient closure of phagophore in the EBSS-treated cells. Analysis of LC3-positive membrane structures using Halo-tagged LC3 autophagosome completion assay showed that predominantly unclosed phagophore rather than closed autophagosome was present in the EBSS-treated MOAP1-deficient cells. The autophagy substrate SQSTM1/p62, which is normally contained within the enclosed autophagosome under EBSS condition, was also highly sensitive to degradation by proteinase K in the absence of MOAP1. MOAP1 binds LC3 and the binding is critically dependent on a LC3-interacting region (LIR) motif detected at its N-terminal region. Re-expression of MOAP1, but not its LC3-binding defective mutant, MOAP1-LIR, in the MOAP1-deficient cells, restored EBSS-induced autophagy. Together, these observations suggest that MOAP1 serves a distinct role in facilitating autophagy through interacting with LC3 to promote efficient phagophore closure during starvation.Abbreviations: CQ: Chloroquine; EBSS: Earle's Balanced Salt Solution; GABARAP: Gamma-Amino Butyric Acid Receptor Associated Protein; IF: Immunofluorescence; IP: Immunoprecipitation; LAMP1: Lysosomal-Associated Membrane Protein 1; LIR: LC3-Interacting Region; MAP1LC3/LC3: Microtubule Associated Protein 1 Light Chain 3; MEF: Mouse Embryonic Fibroblast; MOAP1: Modulator of Apoptosis 1; PE: Phosphatidylethanolamine; PtdIns3K: class III PtdIns3K complex I; PtdIns3P: Phosphatidylinositol-3-phosphate; STX17: Syntaxin 17; ULK1: unc-51 like autophagy activating kinase 1.

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Dataset Information

Nuclear LC3 Associates with Slowly Diffusing Complexes that Survey the Nucleolus.

Publications

Nuclear LC3 Associates with Slowly Diffusing Complexes that Survey the Nucleolus.

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