hNOA1 Interacts with Complex I and DAP3 and Regulates Mitochondrial Respiration and Apoptosis

Abstract

Mitochondria are dynamic organelles that play key roles in metabolism, energy production, and apoptosis. Coordination of these processes is essential to maintain normal cellular functions. Here we characterized hNOA1, the human homologue of AtNOA1 (Arabidopsis thaliana nitric oxide-associated protein 1), a large mitochondrial GTPase. By immunofluorescence, immunoelectron microscopy, and mitochondrial subfractionation, endogenous hNOA1 is localized within mitochondria where it is peripherally associated with the inner mitochondrial membrane facing the mitochondrial matrix. Overexpression and knockdown of hNOA1 led to changes in mitochondrial shape implying effects on mitochondrial dynamics. To identify the interaction partners of hNOA1 and to further understand its cellular functions, we performed immunoprecipitation-mass spectrometry analysis of endogenous hNOA1 from enriched mitochondrial fractions and found that hNOA1 interacts with both Complex I of the electron transport chain and DAP3 (death-associated protein 3), a positive regulator of apoptosis. Knockdown of hNOA1 reduces mitochondrial O 2 consumption ϳ20% in a Complex I-dependent manner, supporting a functional link between hNOA1 and Complex I. Moreover, knockdown of hNOA1 renders cells more resistant to apoptotic stimuli such as ␥-interferon and staurosporine, supporting a role for hNOA1 in regulating apoptosis. Thus, based on its interactions with both Complex I and DAP3, hNOA1 may play a role in mitochondrial respiration and apoptosis.Emerging evidence indicates that mitochondrial metabolism, apoptosis, and dynamics (fission and fusion) are closely intertwined. Apoptosis and changes in metabolism are associated with morphological changes in mitochondria (1, 2). Conversely, when mitochondrial morphology is altered either by mutations or altered expression of mitochondrial fission or fusion proteins such as the dynamin like large G proteins Drp1 and Opa1, the cell's susceptibility to apoptotic agents (3) or ability to generate ATP (4, 5) is altered.Apoptosis is controlled by a diverse range of cell signals, which may originate either extracellularly (extrinsic inducers) or intracellularly (intrinsic inducers), and mitochondria play central roles in both pathways (6). The apoptotic pathways involve a growing list of mitochondria-associated proteins, such as Bad, cytochrome c, Smac, AIF, Bcl-2, and others, most of which are located either on the outer mitochondrial membrane (OMM) 3 or in the intermembrane space (IMS) (7). Recently, proteins of the mitochondrial matrix such as DAP3, have also been shown to be involved in apoptosis (8). DAP3 has been reported to be involved in both ␥-interferon-(9) and tumor necrosis factor-␣-induced (10) apoptosis as well as staurosporine-induced mitochondrial fragmentation (11), but the detailed mechanisms involved remain to be elucidated.Besides their role in apoptosis, much more is known about the functions of mitochondria in respiration and generation of ATP. The electron transport chain in the inner mitochondrial mem...

Quantitative Proteomic and Functional Analysis of Liver Mitochondria from High Fat Diet (HFD) Diabetic Mice

“…"Electron Flow" Respiration Analysis Using the XF24 Flux Analyzer-Methods for respiration analysis of isolated liver mitochondria were adapted from previously described methods (30). All steps were conducted on ice (4°C) unless otherwise stated.…”

“…After centrifugation, 450 l of 1x MAS prewarmed to 37°C was carefully added to each well, and then the plate was equilibrated to room temperature for 5 min before beginning the assay. Subsequent oxygen consumption rate (OCR) analysis of functionally active liver mitochondria was conducted according to the "Electron Flow" assay, with given mix/measure time-course and drug injections, as described previously (30).…”

Mitochondrial Chaperone Trap1 and the Calcium Binding Protein Sorcin Interact and Protect Cells against Apoptosis Induced by Antiblastic Agents

“…Mitochondria were purified using the Qproteome Mitochondria Isolation kit (Qiagen) according to the manufacturer's protocol. Mitochondrial subfractionation was performed according to Tang and colleagues (24). Briefly, 150 μg of mitochondria prepared from HCT-116 cells were swollen with hypotonic medium to break the outer mitochondrial membrane, keeping intact the inner mitochondrial membrane.…”

Nitric Oxide-associated Protein 1 (NOA1) Is Necessary for Oxygen-dependent Regulation of Mitochondrial Respiratory Complexes

“…We also generated a mutant form of mNOA1, which contained an amino acid substitution (K353R) in the P-loop sequence (13,32), to disclose the potential necessity of an intact GTPase domain for mNOA1 function. Transfection of mNOA1 resulted in a strong enhancement of complex IϩIII activity as measured by cytochrome c reduction using NADH as a substrate (corresponding to complex IϩIII activity).…”

“…Guided by the recent description of the mitochondrial proteome (11), we focused on poorly known mitochondrial GTPases. Of these, the mouse homolog of Arabidopsis thaliana nitric oxide-associated protein 1, mNOA1, 3 stood out as a potential factor, which might regulate adaptive responses to changes in oxygen concentration, because mNOA1 is known to control respiratory activity and cell death (12,13) as well as the assembly or stability of ribosomes although its mode of action is enigmatic (14 -16).…”