Regulation of mitochondrial function by ARL2 and its putative effector ELMOD2

描述(申请人提供)：线粒体是重要的和动态的细胞器，为细胞产生能量，线粒体缺陷是几种人类疾病的基础。我们发现小GTP酶ARL2的缺失严重损害了线粒体的功能。具体地说，ARL2活性的丧失会导致线粒体形态、运动性和ATP产生的缺陷。我们还发现，ARL2 GTP酶激活蛋白(GAP)ELMOD2的敲除也会导致形态、运动性和ATP产生的缺陷。由于ARL2和ELMOD2都定位于线粒体基质，可以相互结合，并在基因敲除时产生相同的表型，因此它们可能是重要的线粒体信号通路的成员。此外，ARL2和ELMOD2表型的缺失也导致了OPA1的缺失，OPA1是一种重要的冠状突起调节因子，对线粒体ATP的产生至关重要。因此，我假设ARL2信号通过线粒体基质中的ELMOD2信号是通过调节脊形态来正确维持线粒体的形态、运动性和ATP产生所必需的。在目标1中，我将确定以基质为靶点的ARL2和ELMOD2是否可以修复由于ARL2和ELMOD2基因敲除而导致的线粒体缺陷，这将测试我的模型，即ARL2和ELMOD2从基质中起作用。为了测试ELMOD2是否作为ARL2效应器，我将产生不能与ELMOD2结合的ARL2的点突变，这将确定其线粒体功能是否需要ARL2与ELMOD2结合。我还将使用间隙死亡ELMOD2突变体ELMOD2[R167K]来确定ARL2的ELMOD2间隙活性是否是线粒体功能所必需的。在目标2中，我将使用电子显微镜来确定ARL2和ELMOD2的亚线粒体定位，并验证我的假设，即它们的敲除改变了眉骨的形态。研究这些进化上保守的蛋白质将使我们了解线粒体的正常功能，这可以用于研究许多人类疾病。

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.