Functional characterization of the MENTAL domain
Functional characterization of the MENTAL domain
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DOI：
10.1074/jbc.m500723200
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发表时间：
2005-05-06
影响因子：
4.8
通讯作者：
Tomasetto, C
中科院分区：
文献类型：
--
作者：
Alpy, F;Latchumanan, VK;Tomasetto, C
人转移淋巴结 (MLN) 64 由两个保守区域组成。氨基末端包含一个保守的跨膜 MENTAL（MLN64 NH2 末端）结构域，与一种名为 MENTHO（MLN64 NH2 末端结构域同源物）的独特蛋白质共享，并将该蛋白质靶向晚期内体。羧基末端结构域由暴露于细胞质的胆固醇结合类固醇生成急性调节相关脂质转移结构域组成。 MENTHO 过度表达导致扩大的内体积累。在这项研究中，我们发现 MLN64 过度表达还会诱导扩大内体的形成，这种效应可能是由 MENTAL 结构域介导的。使用体内光胆固醇结合测定，我们发现 MLN64 的 MENTAL 结构域是胆固醇结合结构域。此外，谷胱甘肽 S-转移酶下拉或免疫共沉淀实验表明该结构域介导 MLN64 和 MENTHO 的同源和异源相互作用。在活细胞中，成对的黄色荧光和青色荧光融合蛋白的表达显示出 MENTHO 同源相互作用及其与 MLN64 的相互作用。这些数据表明，在晚期内体膜内，MLN64 和 MENTHO 定义了离散的含胆固醇子结构域。 MENTAL 结构域可能有助于在胆固醇穿梭至细胞质受体之前将胆固醇维持在晚期内涵体的膜上。
Human metastatic lymph node (MLN) 64 is composed of two conserved regions. The amino terminus contains a conserved membrane-spanning MENTAL (MLN64 NH2-terminal) domain shared with an unique protein called MENTHO ( MLN64 NH2-terminal domain homologue) and targets the protein to late endosome. The carboxyl-terminal domain is composed of a cholesterol binding steroidogenic acute regulatory-related lipid transfer domain exposed to the cytoplasm. MENTHO overexpression leads to the accumulation of enlarged endosomes. In this study, we show that MLN64 overexpression also induces the formation of enlarged endosomes, an effect that is probably mediated by the MENTAL domain. Using an in vivo photocholesterol binding assay, we find that the MENTAL domain of MLN64 is a cholesterol binding domain. Moreover, glutathione S-transferase pull-down or co-immunoprecipitation experiments demonstrate that this domain mediates homo- and hetero-interaction of MLN64 and MENTHO. In living cells, the expression of paired yellow fluorescent and cyan fluorescent fusion proteins show MENTHO homo- interaction and its interaction with MLN64. These data indicate that within late-endosomal membranes, MLN64 and MENTHO define discrete cholesterol-containing subdomains. The MENTAL domain might serve to maintain cholesterol at the membrane of late endosomes prior to its shuttle to cytoplasmic acceptor(s).