Solution structure of microtubule-associated protein light chain 3 and identification of its functional subdomains
Solution structure of microtubule-associated protein light chain 3 and identification of its functional subdomains
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DOI：
10.1074/jbc.m413565200
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发表时间：
2005-07-01
影响因子：
4.8
通讯作者：
Kawano, K
中科院分区：
文献类型：
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作者：
Kouno, T;Mizuguchi, M;Kawano, K
微管相关蛋白(MAP)轻链3(Lc3)是酵母Apg8/Aut7/Cvt5(Atg8)的人类同源物，在自噬过程中起着至关重要的作用。MAP-LC3被半胱氨酸蛋白酶切割生成LC3-I，定位于胞浆部分。而LC3-I则通过类E1和类E2酶的作用转化为LC3-II。LC3-II在其C端与磷脂酰乙醇胺共价结合，并与自噬体膜紧密结合。我们确定了LC3-I的溶液结构，发现它分为N-末端和C-末端两个亚域。用光化学诱导的动态核极化技术进行的另外的分析还表明，LC_3-I的N-末端亚区与C-末端亚域的表面接触，并且LC_3-I在溶液中以单一的致密构象存在。此外，在LC_3-I溶液中加入十二烷基磷胆碱主要在C-末端亚区引起化学位移扰动，这意味着这两个亚区对十二烷基磷胆碱胶束具有不同的敏感性。另一方面，N-末端亚区的缺失取消了微管与微管的结合。因此，我们证明了Lc3-I结构的两个亚域具有不同的功能，这表明MAP-Lc3可以作为微管和自噬小体之间的接头蛋白。
Microtubule-associated protein ( MAP) light chain 3 (LC3) is a human homologue of yeast Apg8/Aut7/Cvt5 (Atg8), which is essential for autophagy. MAP-LC3 is cleaved by a cysteine protease to produce LC3-I, which is located in cytosolic fraction. LC3-I, in turn, is converted to LC3-II through the actions of E1- and E2-like enzymes. LC3-II is covalently attached to phosphatidylethanolamine on its C terminus, and it binds tightly to autophagosome membranes. We determined the solution structure of LC3- I and found that it is divided into N- and C-terminal subdomains. Additional analysis using a photochemically induced dynamic nuclear polarization technique also showed that the N- terminal subdomain of LC3- I makes contact with the surface of the C-terminal subdomain and that LC3- I adopts a single compact conformation in solution. Moreover, the addition of dodecylphosphocholine into the LC3- I solution induced chemical shift perturbations primarily in the C-terminal subdomain, which implies that the two subdomains have different sensitivities to dodecylphosphocholine micelles. On the other hand, deletion of the N-terminal subdomain abolished binding of tubulin and microtubules. Thus, we showed that two subdomains of the LC3- I structure have distinct functions, suggesting that MAP-LC3 can act as an adaptor protein between microtubules and autophagosomes.