Olsvik Hallvard L, Lamark Trond, Takagi Kenji, Larsen Kenneth Bowitz, Evjen Gry, Øvervatn Aud, Mizushima Tsunehiro, Johansen Terje
From the Molecular Cancer Research Group, Institute of Medical Biology, University of Tromsø - The Arctic University of Norway, 9037 Tromsø, Norway and.
the Picobiology Institute, Graduate School of Life Science, University of Hyogo, Hyogo 651-2197, Japan.
J Biol Chem. 2015 Dec 4;290(49):29361-74. doi: 10.1074/jbc.M115.686915. Epub 2015 Oct 14.
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.
FYCO1（含FYVE和卷曲螺旋结构域蛋白1）是一种转运衔接蛋白，它可与磷脂酰肌醇3 - 磷酸、Rab7以及微管相关蛋白1轻链3（LC3）结合，介导晚期内体和自噬体沿微管向正端方向运输。我们之前已经表明，FYCO1通过一个包含假定核心LC3相互作用区域（LIR）基序的19个氨基酸序列与LC3B结合。在此，我们表明FYCO1优先与LC3A和LC3B结合。通过基于肽阵列的与LC3B结合的二维突变扫描，我们发现FYCO1含有一个C末端延伸的LIR结构域。我们以1.53 Å的分辨率确定了来自FYCO1的13个氨基酸LIR肽与LC3B之间复合物的晶体结构。通过将结构信息与突变分析相结合，揭示了C末端延伸LIR的基础以及对LC3A/B结合的特异性。FYCO1包含一个9个氨基酸长的F型LIR基序。除了第1位的典型芳香族残基和第3位的疏水残基外，第8位和第9位的酸性残基和疏水残基分别对于与LC3B的有效结合很重要，这解释了C末端的延伸。与LC3A/B结合的特异性是由于FYCO1中的Asp(1285)与LC3B中的His(57)之间的相互作用。为了探究FYCO1的LIR基序的功能意义，我们构建了FYCO1基因敲除细胞，随后用GFP - FYCO1野生型和LIR突变体构建体进行重建。我们的数据表明，在基础条件下，FYCO1需要一个功能性的LIR基序来促进自噬体的有效成熟，而饥饿诱导的自噬则不受影响。
