title: FYCO1 Contains a C-terminally Extended, LC3A/B-preferring LC3-interacting Region (LIR) Motif Required for Efficient Maturation of Autophagosomes during Basal Autophagy.
abstract: FYCO1 (FYVE and coiled-coil protein 1) is a transport adaptor that binds to phosphatidylinositol 3-phosphate, to Rab7, and to LC3 (microtubule-associated protein 1 light chain 3) to mediate transport of late endosomes and autophagosomes along microtubules in the plus end direction. We have previously shown that FYCO1 binds to LC3B via a 19-amino acid sequence containing a putative core LC3-interacting region (LIR) motif. Here, we show that FYCO1 preferentially binds to LC3A and -B. By peptide array-based two-dimensional mutational scans of the binding to LC3B, we found FYCO1 to contain a C-terminally extended LIR domain. We determined the crystal structure of a complex between a 13-amino acid LIR peptide from FYCO1 and LC3B at 1.53 Å resolution. By combining the structural information with mutational analyses, both the basis for the C-terminally extended LIR and the specificity for LC3A/B binding were revealed. FYCO1 contains a 9-amino acid-long F-type LIR motif. In addition to the canonical aromatic residue at position 1 and the hydrophobic residue at position 3, an acidic residue and a hydrophobic residue at positions 8 and 9, respectively, are important for efficient binding to LC3B explaining the C-terminal extension. The specificity for binding to LC3A/B is due to the interaction between Asp(1285) in FYCO1 and His(57) in LC3B. To address the functional significance of the LIR motif of FYCO1, we generated FYCO1 knock-out cells that subsequently were reconstituted with GFP-FYCO1 WT and LIR mutant constructs. Our data show that FYCO1 requires a functional LIR motif to facilitate efficient maturation of autophagosomes under basal conditions, whereas starvation-induced autophagy was unaffected.
authors.affiliation: From the Molecular Cancer Research Group, Institute of Medical Biology, University of Tromsø - The Arctic University of Norway, 9037 Tromsø, Norway and.
authors.forename: Hallvard L
authors.initials: HL
authors.lastname: Olsvik
authors.affiliation: From the Molecular Cancer Research Group, Institute of Medical Biology, University of Tromsø - The Arctic University of Norway, 9037 Tromsø, Norway and.
authors.forename: Trond
authors.initials: T
authors.lastname: Lamark
authors.affiliation: the Picobiology Institute, Graduate School of Life Science, University of Hyogo, Hyogo 651-2197, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Takagi
authors.affiliation: From the Molecular Cancer Research Group, Institute of Medical Biology, University of Tromsø - The Arctic University of Norway, 9037 Tromsø, Norway and.
authors.forename: Kenneth Bowitz
authors.initials: KB
authors.lastname: Larsen
authors.affiliation: From the Molecular Cancer Research Group, Institute of Medical Biology, University of Tromsø - The Arctic University of Norway, 9037 Tromsø, Norway and.
authors.forename: Gry
authors.initials: G
authors.lastname: Evjen
authors.affiliation: From the Molecular Cancer Research Group, Institute of Medical Biology, University of Tromsø - The Arctic University of Norway, 9037 Tromsø, Norway and.
authors.forename: Aud
authors.initials: A
authors.lastname: Øvervatn
authors.affiliation: the Picobiology Institute, Graduate School of Life Science, University of Hyogo, Hyogo 651-2197, Japan.
authors.forename: Tsunehiro
authors.initials: T
authors.lastname: Mizushima
authors.affiliation: From the Molecular Cancer Research Group, Institute of Medical Biology, University of Tromsø - The Arctic University of Norway, 9037 Tromsø, Norway and terje.johansen@uit.no.
authors.forename: Terje
authors.identifier: http://orcid.org/0000-0003-1451-9578
authors.initials: T
authors.lastname: Johansen
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001343:Autophagy; D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010588:Phagosomes / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; C460141:FYCO1 protein, human; C477012:MAP1LC3A protein, human; C502070:MAP1LC3B protein, human; D008869:Microtubule-Associated Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins
keywords: FYCO1; LC3; LIR; autophagy; crystal structure; protein degradation; protein-protein interaction; vesicles