Selective Transport of α-Mannosidase by Autophagic Pathways STRUCTURAL BASIS FOR CARGO RECOGNITION BY Atg19 AND Atg34
Selective Transport of α-Mannosidase by Autophagic Pathways STRUCTURAL BASIS FOR CARGO RECOGNITION BY Atg19 AND Atg34
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DOI：
10.1074/jbc.m110.143545
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发表时间：
2010-09-24
影响因子：
4.8
通讯作者：
Inagaki, Fuyuhiko
中科院分区：
文献类型：
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作者：
Watanabe, Yasunori;Noda, Nobuo N.;Inagaki, Fuyuhiko
在酿酒酵母中，氨肽酶I（prApe 1）和α-甘露糖苷酶（Ams 1）的前体形式在营养条件下通过细胞质至液泡靶向途径选择性地转运至液泡，并且在饥饿条件下通过自噬转运至液泡。Atg 19通过将Ams 1和prApe 1连接到Atg 8和Atg 11而在这些过程中发挥核心作用。然而，很少有人知道货物识别的Atg 19的分子机制。在这里，我们报告的结构和功能分析的Atg 19和它的paradigm，Atg 34。在Atg 19的C-末端区域中鉴定了蛋白酶抗性结构域，该结构域在Atg 34中也是保守的。体外下拉试验表明，Atg 19和Atg 34的C-末端结构域负责Ams 1结合;这些结构域在下文中被称为Ams 1结合结构域（ABD）。在表达Atg 19（Δ ABD）的atg 19 Δ atg 34 Δ细胞中，Ams 1的转运被阻断，但PrApe 1未被阻断，表明ABD是Ams 1转运所特异性需要的。然后，我们使用NMR光谱确定了Atg 19和Atg 34的ABD的溶液结构。两种ABD结构都具有典型的免疫球蛋白折叠，其由八条β链组成，其中高度保守的环聚集在折叠的一侧。这些事实，连同突变分析的结果，表明ABD承认Ams 1使用这些保守的环。
In the yeast Saccharomyces cerevisiae, a precursor form of aminopeptidase I (prApe1) and alpha-mannosidase (Ams1) are selectively transported to the vacuole through the cytoplasm-to-vacuole targeting pathway under vegetative conditions and through autophagy under starvation conditions. Atg19 plays a central role in these processes by linking Ams1 and prApe1 to Atg8 and Atg11. However, little is known about the molecular mechanisms of cargo recognition by Atg19. Here, we report structural and functional analyses of Atg19 and its paralog, Atg34. A protease-resistant domain was identified in the C-terminal region of Atg19, which was also conserved in Atg34. In vitro pulldown assays showed that the C-terminal domains of both Atg19 and Atg34 are responsible for Ams1 binding; these domains are hereafter referred to as Ams1-binding domains (ABDs). The transport of Ams1, but not prApe1, was blocked in atg19 Delta atg34 Delta cells expressing Atg19(Delta ABD), indicating that ABD is specifically required for Ams1 transport. We then determined the solution structures of the ABDs of Atg19 and Atg34 using NMR spectroscopy. Both ABD structures have a canonical immunoglobulin fold consisting of eight beta-strands with highly conserved loops clustered at one side of the fold. These facts, together with the results of a mutational analysis, suggest that ABD recognizes Ams1 using these conserved loops.