Department of Neurology, University of Michigan, Ann Arbor, Michigan 48109, USA.
J Biol Chem. 2013 Jun 14;288(24):17145-55. doi: 10.1074/jbc.M113.463406. Epub 2013 Apr 26.
The functional diversity of deubiquitinating enzymes (DUBs) is not well understood. The MJD family of DUBs consists of four cysteine proteases that share a catalytic "Josephin" domain. The family is named after the DUB ATXN3, which causes the neurodegenerative disease Machado-Joseph disease. The two closely related Josephin domain-containing (JosD) proteins 1 and 2 consist of little more than the Josephin domain. To gain insight into the properties of Josephin domains, we investigated JosD1 and JosD2. JosD1 and JosD2 were found to differ fundamentally in many respects. In vitro, only JosD2 can cleave ubiquitin chains. In contrast, JosD1 cleaves ubiquitin chains only after it is monoubiquitinated, a form of posttranslational-dependent regulation shared with ATXN3. A significant fraction of JosD1 is monoubiquitinated in diverse mouse tissues. In cell-based studies, JosD2 localizes to the cytoplasm whereas JosD1 preferentially localizes to the plasma membrane, particularly when ubiquitinated. The membrane occupancy by JosD1 suggests that it could participate in membrane-dependent events such as cell motility and endocytosis. Indeed, time-lapse imaging revealed that JosD1 enhances membrane dynamics and cell motility. JosD1 also influences endocytosis in cultured cells by increasing the uptake of endocytic markers of macropinocytosis while decreasing those for clathrin- and caveolae-mediated endocytosis. Our results establish that two closely related DUBs differ markedly in activity and function and that JosD1, a membrane-associated DUB whose activity is regulated by ubiquitination, helps regulate membrane dynamics, cell motility, and endocytosis.
去泛素化酶（DUBs）的功能多样性尚未得到很好的理解。MJD 家族的 DUBs 由四个半胱氨酸蛋白酶组成，它们共享一个催化的“Josephin”结构域。该家族以 DUB ATXN3 命名，后者导致神经退行性疾病 Machado-Joseph 病。两个密切相关的含有 Josephin 结构域的（JosD）蛋白 1 和 2 几乎只包含 Josephin 结构域。为了深入了解 Josephin 结构域的特性，我们研究了 JosD1 和 JosD2。发现 JosD1 和 JosD2 在许多方面存在根本差异。在体外，只有 JosD2 可以切割泛素链。相比之下，JosD1 仅在单泛素化后才能切割泛素链，这是一种与 ATXN3 共享的翻译后依赖性调节形式。在不同的小鼠组织中，JosD1 的很大一部分被单泛素化。在基于细胞的研究中，JosD2 定位于细胞质，而 JosD1 优先定位于质膜，尤其是在泛素化时。JosD1 占据质膜的位置表明它可能参与膜依赖事件，如细胞运动和内吞作用。事实上，延时成像显示 JosD1 增强了膜动力学和细胞运动。JosD1 还通过增加大胞饮作用的内吞标记物的摄取，同时减少网格蛋白和小窝介导的内吞作用的摄取，来影响培养细胞中的内吞作用。我们的结果表明，两种密切相关的 DUBs 在活性和功能上有显著差异，并且 JosD1 是一种膜相关的 DUB，其活性受泛素化调节，有助于调节膜动力学、细胞运动和内吞作用。
