Regulation of mitochondrial function by ARL2 and its putative effector ELMOD2

描述（由申请人提供）：线粒体是重要的动态细胞器，为细胞产生能量，线粒体缺陷是几种人类疾病的基础。我们已经发现，小GTTARL2的丢失严重损害线粒体功能。具体而言，ARL2活性的丧失导致线粒体形态、运动性和ATP产生的缺陷。我们还发现，ARL2 GT3激活蛋白（GAP）ELMOD 2的敲低也会导致形态学、运动性和ATP产生的缺陷。由于ARL2和ELMOD 2都定位于线粒体基质，可以相互结合，并在敲低后产生相同的表型，因此它们可能是重要的线粒体信号传导途径的成员。此外，ARL2和ELMOD 2表型的丧失导致了OPA 1的丧失，OPA 1是嵴的重要调节因子，对线粒体ATP的产生至关重要。因此，我推测，ARL2信号通过ELMOD 2在线粒体基质中所需的适当维护线粒体形态，运动性，ATP的生产，通过调节嵴形态。在目标1中，我将确定基质靶向的ARL2和ELMOD 2是否可以挽救由ARL2和ELMOD 2敲低引起的线粒体缺陷，这将测试我的模型，即ARL2和ELMOD 2从基质内起作用。为了测试ELMOD 2是否作为ARL2效应物，我将产生不能结合ELMOD 2的ARL2的点突变体，这将确定ARL2与ELMOD 2的结合是否是其线粒体功能所需的。我还将使用GAP死亡ELMOD 2突变体ELMOD 2 [R167K]来确定ARL2的ELMOD 2 GAP活性是否是线粒体功能所需的。在目标2中，我将使用电子显微镜来确定ARL2和ELMOD 2的亚线粒体定位，并测试我的假设，即它们的敲除改变嵴形态。研究这些进化上保守的蛋白质将告诉我们适当的线粒体功能，这可用于许多人类疾病的研究。

DESCRIPTION (provided by applicant): Mitochondria are important and dynamic organelles that produce energy for the cell, and mitochondrial defects underlie several human diseases. We have found that loss of the small GTPase ARL2 severely impairs mitochondrial function. Specifically, loss of ARL2 activity causes defects in mitochondrial morphology, motility, and ATP production. We have also found that knockdown of an ARL2 GTPase activating protein (GAP), ELMOD2, also causes defects in morphology, motility, and ATP production. Because both ARL2 and ELMOD2 localize to the mitochondrial matrix, can bind to each other, and produce the same phenotypes upon knockdown, they are likely members of an important mitochondrial signaling pathway. Additionally, loss of ARL2 and ELMOD2 phenocopy the loss of OPA1, an important regulator of cristae, which are critical for mitochondrial ATP production. Therefore, I hypothesize that ARL2 signaling through ELMOD2 in the mitochondrial matrix is required for proper maintenance of mitochondrial morphology, motility, and ATP production, through regulation of cristae morphology. In Aim 1 I will determine if matrix-targeted ARL2 and ELMOD2 can rescue the mitochondrial defects resulting from ARL2 and ELMOD2 knockdown, which will test my model that ARL2 and ELMOD2 act from within the matrix. To test if ELMOD2 acts as an ARL2 effector, I will generate point mutants of ARL2 that cannot bind ELMOD2, which will determine if ARL2 binding to ELMOD2 is required for its mitochondrial functions. I will also use a GAP dead ELMOD2 mutant, ELMOD2[R167K], to determine if ELMOD2 GAP activity for ARL2 is required for mitochondrial function. In Aim 2 I will use electron microscopy to determine the sub-mitochondrial localization of ARL2 and ELMOD2, and test my hypothesis that their knockdown alters cristae morphology. Studying these evolutionarily conserved proteins will inform us about proper mitochondrial function, which can be used for the study of many human diseases.