FYCO1 Contains a C-terminally Extended, LC3A/B-preferring LC3-interacting Region (LIR) Motif Required for Efficient Maturation of Autophagosomes during Basal Autophagy
FYCO1 Contains a C-terminally Extended, LC3A/B-preferring LC3-interacting Region (LIR) Motif Required for Efficient Maturation of Autophagosomes during Basal Autophagy
复制标题
DOI：
10.1074/jbc.m115.686915
复制
发表时间：
2015-12-04
影响因子：
4.8
通讯作者：
Johansen, Terje
中科院分区：
文献类型：
--
作者：
Olsvik, Hallvard L.;Lamark, Trond;Johansen, Terje
FYCO1（FYVE 和卷曲螺旋蛋白 1）是一种转运接头，可与 3-磷酸磷脂酰肌醇、Rab7 和 LC3（微管相关蛋白 1 轻链 3）结合，介导晚期核内体和自噬体沿微管正端方向的转运。我们之前已经证明，FYCO1 通过包含假定的核心 LC3 相互作用区 (LIR) 基序的 19 个氨基酸序列与 LC3B 结合。在这里，我们表明 FYCO1 优先结合 LC3A 和 -B。通过基于肽阵列的二维突变扫描与 LC3B 的结合，我们发现 FYCO1 包含 C 端延伸的 LIR 结构域。我们以 1.53 埃的分辨率确定了 FYCO1 的 13 个氨基酸 LIR 肽和 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 端延伸 LIR 的基础和 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。除了位置 1 的典型芳香残基和位置 3 的疏水残基之外，位置 8 和位置 9 的酸性残基和疏水残基分别对于有效结合 LC3B 解释 C 末端延伸也很重要。与 LC3A/B 结合的特异性是由于 FYCO1 中的 Asp1285 和 LC3B 中的 His(57) 之间的相互作用。为了解决 FYCO1 LIR 基序的功能意义，我们生成了 FYCO1 敲除细胞，随后用 GFP-FYCO1 WT 和 LIR 突变体构建体进行重组。我们的数据表明，FYCO1 需要功能性 LIR 基序来促进自噬体在基础条件下有效成熟，而饥饿诱导的自噬不受影响。
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 angstrom 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 Asp1285 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.