Differential Roles of C-terminal Eps15 Homology Domain Proteins as Vesiculators and Tubulators of Recycling Endosomes
Differential Roles of C-terminal Eps15 Homology Domain Proteins as Vesiculators and Tubulators of Recycling Endosomes
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DOI：
10.1074/jbc.m113.488627
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发表时间：
2013-10-18
影响因子：
4.8
通讯作者：
Caplan, Steve
中科院分区：
文献类型：
--
作者：
Cai, Bishuang;Giridharan, Sai Srinivas Panapakkam;Caplan, Steve
背景：管状再循环内体的囊泡化对于受体和脂质向质膜的再循环是必不可少的。结果：一种新的囊泡形成实验被用来证明内吞调节蛋白在囊泡形成中的作用。结论：EHD家族蛋白在囊泡形成和肾小管再循环内体的形成中起重要作用。重要性：这提供了第一个直接的证据，差异EHD功能的泡和tuberosis.Endocytic回收涉及返回的膜和受体的质膜后，他们的内化到细胞。再循环通常发生在位于核周区域的一系列囊泡和管状膜，统称为内吞再循环室。在该隔室内，受体被分选到随后经历囊泡化的管状延伸中，允许运输囊泡沿着沿着微管移动并返回到细胞表面，在那里它们最终经历与质膜的融合。最近的研究已经导致假设，C-末端Eps 15同源结构域（EHD）ATP酶蛋白参与囊泡形成过程。在这里，我们解决四个EHD蛋白的功能作用。我们开发了一种新的半透化细胞系统，其中添加纯化的EHD蛋白质来重建囊泡形成，使我们能够评估每种蛋白质囊泡化MICAL-L1修饰的管状再循环内体（TREs）的能力。使用此测定，我们表明，EHD 1囊泡膜，增强TRE生成EHD 1耗尽后观察到一致。EHD 4在TRE囊泡形成中起类似于EHD 1的作用，而EHD 2尽管能够在半透化细胞中囊泡化TRE，但在体内不能这样做。令人惊讶的是，EHD 3的加入导致我们的半透化细胞系统中的内吞膜的微管化，这与EHD 3耗尽后观察到的微管化的缺乏一致。我们的新的囊泡形成试验和体外电子显微镜分析，结合体内数据，提供的证据表明，EHD 1和EHD 4的功能主要是在TRE膜囊泡形成，而EHD 3是一种膜微管蛋白。
Background: Vesiculation of tubular recycling endosomes is essential for the recycling of receptors and lipids to the plasma membrane. Results: A novel vesiculation assay was used to demonstrate a role for endocytic regulatory proteins in vesiculation. Conclusion: EHD family proteins play significant roles in both the vesiculation and generation of tubular recycling endosomes. Significance: This provides the first direct evidence of differential EHD function in vesiculation and tubulation.Endocytic recycling involves the return of membranes and receptors to the plasma membrane following their internalization into the cell. Recycling generally occurs from a series of vesicular and tubular membranes localized to the perinuclear region, collectively known as the endocytic recycling compartment. Within this compartment, receptors are sorted into tubular extensions that later undergo vesiculation, allowing transport vesicles to move along microtubules and return to the cell surface where they ultimately undergo fusion with the plasma membrane. Recent studies have led to the hypothesis that the C-terminal Eps15 homology domain (EHD) ATPase proteins are involved in the vesiculation process. Here, we address the functional roles of the four EHD proteins. We developed a novel semipermeabilized cell system in which addition of purified EHD proteins to reconstitute vesiculation allows us to assess the ability of each protein to vesiculate MICAL-L1-decorated tubular recycling endosomes (TREs). Using this assay, we show that EHD1 vesiculates membranes, consistent with enhanced TRE generation observed upon EHD1 depletion. EHD4 serves a role similar to that of EHD1 in TRE vesiculation, whereas EHD2, despite being capable of vesiculating TREs in the semipermeabilized cells, fails to do so in vivo. Surprisingly, the addition of EHD3 causes tubulation of endocytic membranes in our semipermeabilized cell system, consistent with the lack of tubulation observed upon EHD3 depletion. Our novel vesiculation assay and in vitro electron microscopy analysis, combined with in vivo data, provide evidence that the functions of both EHD1 and EHD4 are primarily in TRE membrane vesiculation, whereas EHD3 is a membrane-tubulating protein.