Guerra Damian, Chapiro Sonia M, Pratelli Réjane, Yu Shi, Jia Weitao, Leary Julie, Pilot Guillaume, Callis Judy
From the Department of Molecular and Cellular Biology, University of California, Davis, Davis, California 95616 and.
Department of Plant Pathology, Physiology, and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061.
J Biol Chem. 2017 Mar 3;292(9):3827-3840. doi: 10.1074/jbc.M116.766469. Epub 2017 Jan 18.
Intercellular amino acid transport is essential for the growth of all multicellular organisms, and its dysregulation is implicated in developmental disorders. By an unknown mechanism, amino acid efflux is stimulated in plants by overexpression of a membrane-localized protein (LUTAMINE MPER 1 (GDU1)) that requires a ubiquitin ligase ( (). Here we further explore the physiological consequences of the interaction between these two proteins. LOG2 ubiquitin ligase activity is necessary for GDU1-dependent tolerance to exogenous amino acids, and LOG2 self-ubiquitination was markedly stimulated by the GDU1 cytosolic domain, suggesting that GDU1 functions as an adaptor or coactivator of amino acid exporter(s). However, other consequences more typical of a ligase-substrate relationship are observed: disruption of the gene increased the half-life of GDU1, mass spectrometry confirmed that LOG2 ubiquitinates GDU1 at cytosolic lysines, and GDU1 protein levels decreased upon co-expression with active, but not enzymatically inactive LOG2. Altogether these data indicate LOG2 negatively regulates GDU1 protein accumulation by a mechanism dependent upon cytosolic GDU1 lysines. Although GDU1-lysine substituted protein exhibited diminished ubiquitination, overexpression of GDU1 lysine mutants still conferred amino acid tolerance in a LOG2-dependent manner, consistent with GDU1 being both a substrate and facilitator of LOG2 function. From these data, we offer a model in which GDU1 activates LOG2 to stimulate amino acid export, a process that could be negatively regulated by GDU1 ubiquitination and LOG2 self-ubiquitination.
细胞间氨基酸转运对于所有多细胞生物的生长至关重要，其失调与发育障碍有关。通过一种未知机制，植物中一种膜定位蛋白（谷氨酰胺转运蛋白1（GDU1））的过表达会刺激氨基酸外流，而该蛋白需要一种泛素连接酶（）。在这里，我们进一步探讨这两种蛋白相互作用的生理后果。LOG2泛素连接酶活性对于GDU1依赖的对外源氨基酸的耐受性是必需的，并且GDU1胞质结构域显著刺激了LOG2的自我泛素化，这表明GDU1作为氨基酸输出蛋白的衔接子或共激活因子发挥作用。然而，还观察到了其他更典型的连接酶 - 底物关系的后果：基因的破坏增加了GDU1的半衰期，质谱证实LOG2在胞质赖氨酸处使GDU1泛素化，并且与有活性但无酶活性的LOG2共表达时，GDU1蛋白水平降低。总之，这些数据表明LOG2通过依赖于胞质GDU1赖氨酸的机制负向调节GDU1蛋白积累。尽管GDU1赖氨酸替代蛋白的泛素化减少，但GDU1赖氨酸突变体的过表达仍以LOG2依赖的方式赋予氨基酸耐受性，这与GDU1既是LOG2功能的底物又是促进因子一致。基于这些数据，我们提出了一个模型，其中GDU1激活LOG2以刺激氨基酸输出，这一过程可能受到GDU1泛素化和LOG2自我泛素化的负向调节。
