Mitochondrial calcium (Ca2+) uptake has been studied for over five decades, with crucial insights into its underlying mechanisms enabled by development of the chemi-osmotic hypothesis and appreciation of the considerable voltage present across the inner mitochondrial membrane (ΔΨm) generated by proton pumping by the respiratory chain (Carafoli, 1987; Nicholls, 2005). However, the molecules that regulate mitochondrial Ca2+ uptake have only recently been identified (Jiang et. al., 2009; Perocchi et. al., 2010) and further work was needed to clarify how these molecules regulate mitochondrial Ca2+ uptake. Leucine Zipper EF hand containing Transmembrane Protein 1 (LETM1) acts as a regulator of mitochondrial Ca2+ uptake distinct from the mitochondrial Ca2+ uniporter (MCU) pathway (Jiang et. al., 2009). However, a controversy exists regarding the function of LETM1 (Nowikovsky et. al., 2004). Therefore, I asked if LETM1 played a role in mitochondrial Ca2+ uptake and if LETM1 regulated cellular bioenergetics and basal autophagy. To further characterize mitochondrial calcium uptake, we asked how Mitochondrial Calcium Uptake 1 (MICU1) regulates MCU activity by quantifying basal mitochondrial Ca2+ and MCU uptake rates in MICU1 ablated cells. The following work characterizes the molecules that regulate mitochondrial Ca2+ uptake and their mechanistic function on decoding calcium signals. Since LETM1 is the Ca2+/H+ antiporter, I hypothesize that alterations in LETM1 expression and activity will decrease mitochondrial Ca2+ uptake and will result in impaired mitochondrial bioenergetics. As a regulator of free intracellular Ca2+, mitochondrial Ca2+ uptake and the orchestra of its regulatory molecules have been implicated in many human diseases. Mitochondria act both upstream by regulating cytosolic Ca2+ concentration and as downstream effectors that respond to Ca2+ signals. Recently, LETM1 was proposed as a mitochondrial Ca2+/H+ antiporter (Jiang et. al., 2009); however characterization of the functional role of LETM1-mediated Ca2+ transfer remained unstudied. Therefore the specific aims of this project were to determine how LETM1 regulates Ca2+ homeostasis and bioenergetics under physiological settings. Secondly, this project aimed to characterize how LETM1-dependent Ca2+ signaling regulates ROS production and autophagy. The data presented here confirmed that LETM1 knockdown significantly impairs mitochondrial Ca2+ uptake. Furthermore, in-depth approaches including either deletion of EF-hand or mutation of critical EF-hand residues (D676A D688KLETM1) impaired histamine (GPCR agonist)-induced mitochondrial Ca2+ uptake. Knockdown of LETM1 resulted in bioenergetic collapse and promoted LC3-positive multilamellar vesicle formation, indicative of autophagy induction. Interestingly, knockdown of LETM1 significantly reduced complex IV but not complex I and complex II-mediated oxygen consumption rate (OCR). In contrast, cellular NADH and mitochondrial membrane potential (ΔΨm) were unaltered in both control and LETM1 knockdown cells. LETM1 has been implicated in formation of the supercomplexes of the electron transport chain (Tamai et. al., 2008). In support, these studies show that LETM1 knockdown results in increased reactive oxygen species (ROS) production. These results for the first time demonstrate that LETM1 controls cellular bioenergetics through regulation of mitochondrial Ca2+ and ROS. MICU1 was identified as an essential regulator of the mitochondrial Ca2+ uniporter (Perocchi et. al., 2010). Therefore, this project specifically aimed to determine how MICU1 regulates the mitochondrial Ca2+ uniporter. Interestingly, the data presented here suggest that MICU1 is not necessary for uniporter activity. Instead, loss of MICU1 caused mitochondria to constitutively load Ca2+ at rest which resulted in a host of cellular phenotypes. This result led to further questions on how MICU1 knockdown affects cellular bioenergetics and if MICU1 is essential for cell survival under stress. MICU1 ablation influenced pyruvate dehydrogenase activity and ROS production. Subsequent investigations demonstrated that increased basal ROS left cells poised to ceramide-induced cell death thereby suggesting the role of MICU1 in cell survival. Collectively, the data presented here show that MICU1 is necessary to control constitutive mitochondrial Ca2+ uptake during rest. This work demonstrates that LETM1 regulates a distinct mode of mitochondrial Ca2+ uptake pathway whereas MICU1 controls mitochondrial Ca2+ uniporter activity. Further studies are required to uncover the potential role of these two mitochondrial-resident Ca2+ regulators in health and disease. / Biochemistry