Mitochondrial phosphatidylethanolamine synthesis affects mitochondrial energy metabolism and quiescence entry through attenuation of Snf1/AMPK signaling in yeast.

The Ups2-Mdm35 complex mediates intramitochondrial phosphatidylserine (PS) transport to facilitate mitochondrial phosphatidylethanolamine (PE) synthesis. In the present study, we found that ups2∆ yeast showed increased mitochondrial ATP production and enhanced quiescence (G0) entry in the post-diauxic shift phase. Transcriptomic and biochemical analyses revealed that the depletion of Ups2 leads to overactivation of the yeast AMPK homolog Snf1. Inactivation of Snf1 by depletion of an Snf1-activating kinase, Sak1 canceled the changes in mitochondrial ATP production and quiescence entry observed in ups2∆ cells. Furthermore, among the factors regulated by Snf1, upregulation of pyruvate carboxylase, Pyc1 and downregulation of acetyl-CoA carboxylase, Acc1, respectively, were sufficient to increase mitochondrial ATP production and quiescence entry. These results suggested that a normal PE synthesis mediated by Ups2-Mdm35 complex attenuates Snf1/AMPK activity, and that Snf1-mediated regulation of carbon metabolisms has great impacts on mitochondrial energy metabolism and quiescence entry. We also found that depletion of Ups2 together with the cell-cycle regulators Whi5 and Whi7, functional orthologs of the Rb1 tumor suppressor, caused a synthetic growth defect in yeast. Similarly, knockdown of PRELID3b, the human homolog of Ups2, decreased the viability of Rb1-deficient breast cancer cells, suggesting that PRELID3b is a potential target for cancer therapy.

Topology of phosphatidylserine synthase 1 in the endoplasmic reticulum membrane.

Phosphatidylserine (PS) synthase 1 (PSS1) of mammalian cells is a multiple membrane-spanning protein of the endoplasmic reticulum (ER) and regulated by inhibition with the product PS. Alanine-scanning mutagenesis of PSS1 has revealed eight amino acid residues as those crucial for its activity and six as those important for its regulation. Furthermore, three missense mutations in the human PSS1 gene, which lead to regulatory dysfunctions of PSS1 and are causative of Lenz-Majewski syndrome, have been identified. In this study, we investigated the membrane topology of PSS1 by means of epitope insertion and immunofluorescence. According to a 10-transmembrane segment model supported by topology analysis of PSS1, all the 8 amino acid residues crucial for the enzyme activity were localized to the luminal side of the lipid bilayer or the lumen of the ER, whereas all the 9 amino acid residues involved in the enzyme regulation were localized to the cytosol or the cytoplasmic side of the lipid bilayer of the ER. This localization of the functional amino acid residues suggests that PSS1 is regulated by inhibition with PS in the cytoplasmic leaflet of the ER membrane and synthesizes PS at the luminal leaflet.

Structural Basis of Mitochondrial Scaffolds by Prohibitin Complexes: Insight into a Role of the Coiled-Coil Region.

The coiled-coil motif mediates subunit oligomerization and scaffolding and underlies several fundamental biologic processes. Prohibitins (PHBs), mitochondrial inner membrane proteins involved in mitochondrial homeostasis and signal transduction, are predicted to have a coiled-coil motif, but their structural features are poorly understood. Here we solved the crystal structure of the heptad repeat (HR) region of PHB2 at 1.7-Å resolution, showing that it assembles into a dimeric, antiparallel coiled-coil with a unique negatively charged area essential for the PHB interactome in mitochondria. Disruption of the HR coiled-coil abolishes well-ordered PHB complexes and the mitochondrial tubular networks accompanying PHB-dependent signaling. Using a proximity-dependent biotin identification (BioID) technique in live cells, we mapped a number of mitochondrial intermembrane space proteins whose association with PHB2 relies on the HR coiled-coil region. Elucidation of the PHB complex structure in mitochondria provides insight into essential PHB interactomes required for mitochondrial dynamics as well as signal transduction.

Porin Associates with Tom22 to Regulate the Mitochondrial Protein Gate Assembly.

Mitochondria import nearly all of their resident proteins from the cytosol, and the TOM complex functions as their entry gate. The TOM complex undergoes a dynamic conversion between the majority population of a three-channel gateway ("trimer") and the minor population that lacks Tom22 and has only two Tom40 channels ("dimer"). Here, we found that the porin Por1 acts as a sink to bind newly imported Tom22. This Por1 association thereby modulates Tom22 integration into the TOM complex, guaranteeing formation of the functional trimeric TOM complex. Por1 sequestration of Tom22 dissociated from the trimeric TOM complex also enhances the dimeric TOM complex, which is preferable for the import of TIM40/MIA-dependent proteins into mitochondria. Furthermore, Por1 appears to contribute to cell-cycle-dependent variation of the functional trimeric TOM complex by chaperoning monomeric Tom22, which arises from the cell-cycle-controlled variation of phosphorylated Tom6.

Porin proteins have critical functions in mitochondrial phospholipid metabolism in yeast.

Mitochondrial synthesis of cardiolipin (CL) and phosphatidylethanolamine requires the transport of their precursors, phosphatidic acid and phosphatidylserine, respectively, to the mitochondrial inner membrane. In yeast, the Ups1-Mdm35 and Ups2-Mdm35 complexes transfer phosphatidic acid and phosphatidylserine, respectively, between the mitochondrial outer and inner membranes. Moreover, a Ups1-independent CL accumulation pathway requires several mitochondrial proteins with unknown functions including Mdm31. Here, we identified a mitochondrial porin, Por1, as a protein that interacts with both Mdm31 and Mdm35 in budding yeast (Saccharomyces cerevisiae). Depletion of the porins Por1 and Por2 destabilized Ups1 and Ups2, decreased CL levels by ∼90%, and caused loss of Ups2-dependent phosphatidylethanolamine synthesis, but did not affect Ups2-independent phosphatidylethanolamine synthesis in mitochondria. Por1 mutations that affected its interactions with Mdm31 and Mdm35, but not respiratory growth, also decreased CL levels. Using HeLa cells, we show that mammalian porins also function in mitochondrial CL metabolism. We conclude that yeast porins have specific and critical functions in mitochondrial phospholipid metabolism and that porin-mediated regulation of CL metabolism appears to be evolutionarily conserved.

Cooperative function of Fmp30, Mdm31, and Mdm32 in Ups1-independent cardiolipin accumulation in the yeast Saccharomyces cerevisiae.

Cardiolipin (CL) is synthesized from phosphatidic acid (PA) through a series of enzymatic reactions occurring at the mitochondrial inner membrane (MIM). Ups1-Mdm35 mediates PA transfer from the mitochondrial outer membrane (MOM) to the MIM in the yeast Saccharomyces cerevisiae. Deletion of UPS1 leads to a ~80% decrease in the cellular CL level. However, the CL accumulation in ups1∆ cells is enhanced by the depletion of Ups2, which forms a protein complex with Mdm35 and mediates phosphatidylserine (PS) transfer from the MOM to the MIM for phosphatidylethanolamine (PE) synthesis by a PS decarboxylase, Psd1. In this study, we found that the accumulation of CL in ups1∆ cells was enhanced by deletion of not only UPS2, but also PSD1 and CHO1 encoding a PS synthase, suggesting that low PE levels in mitochondria were relevant to the enhancement of CL accumulation in ups1∆ cells. Furthermore, the Ups1-independent and low-level PE-enhanced CL accumulation was shown to depend on the functions of FMP30, MDM31, and MDM32. In addition, the physical interactions of Fmp30 with Mdm31 and Mdm32 were revealed. Thus, when the mitochondrial PE level is reduced, Fmp30, Mdm31, and Mdm32 seem to function cooperatively for the accumulation of CL in a UPS1-independent manner.

Phosphatidylserine transport by Ups2-Mdm35 in respiration-active mitochondria.

Phosphatidylethanolamine (PE) is an essential phospholipid for mitochondrial functions and is synthesized mainly by phosphatidylserine (PS) decarboxylase at the mitochondrial inner membrane. In Saccharomyces cerevisiae, PS is synthesized in the endoplasmic reticulum (ER), such that mitochondrial PE synthesis requires PS transport from the ER to the mitochondrial inner membrane. Here, we provide evidence that Ups2-Mdm35, a protein complex localized at the mitochondrial intermembrane space, mediates PS transport for PE synthesis in respiration-active mitochondria. UPS2- and MDM35-null mutations greatly attenuated conversion of PS to PE in yeast cells growing logarithmically under nonfermentable conditions, but not fermentable conditions. A recombinant Ups2-Mdm35 fusion protein exhibited phospholipid-transfer activity between liposomes in vitro. Furthermore, UPS2 expression was elevated under nonfermentable conditions and at the diauxic shift, the metabolic transition from glycolysis to oxidative phosphorylation. These results demonstrate that Ups2-Mdm35 functions as a PS transfer protein and enhances mitochondrial PE synthesis in response to the cellular metabolic state.

Drp1-dependent mitochondrial fission via MiD49/51 is essential for apoptotic cristae remodeling.

Mitochondrial fission facilitates cytochrome c release from the intracristae space into the cytoplasm during intrinsic apoptosis, although how the mitochondrial fission factor Drp1 and its mitochondrial receptors Mff, MiD49, and MiD51 are involved in this reaction remains elusive. Here, we analyzed the functional division of these receptors with their knockout (KO) cell lines. In marked contrast to Mff-KO cells, MiD49/MiD51-KO and Drp1-KO cells completely resisted cristae remodeling and cytochrome c release during apoptosis. This phenotype in MiD49/51-KO cells, but not Drp1-KO cells, was completely abolished by treatments disrupting cristae structure such as OPA1 depletion. Unexpectedly, OPA1 oligomers generally thought to resist cytochrome c release by stabilizing the cristae structure were similarly disassembled in Drp1-KO and MiD49/51-KO cells, indicating that disassembly of OPA1 oligomers is not directly linked to cristae remodeling for cytochrome c release. Together, these results indicate that Drp1-dependent mitochondrial fission through MiD49/MiD51 regulates cristae remodeling during intrinsic apoptosis.

VID22 is required for transcriptional activation of the PSD2 gene in the yeast Saccharomyces cerevisiae.

Phosphatidylethanolamine (PE) in the yeast Saccharomyces cerevisiae is synthesized through decarboxylation of phosphatidylserine (PS), catalysed by PS decarboxylase 1 (Psd1p) and 2 (Psd2p) and the cytidine 5'-diphosphate (CDP)-ethanolamine (CDP-Etn) pathway. PSD1 null (psd1Δ) and PSD2 null (psd2Δ) mutants are viable in a synthetic minimal medium, but a psd1Δ psd2Δ double mutant exhibits Etn auxotrophy, which is incorporated into PE through the CDP-Etn pathway. We have previously shown that psd1Δ is synthetic lethal with deletion of VID22 (vid22Δ) [Kuroda et al. (2011) Mol. Microbiol. 80: , 248-265]. In the present study, we found that vid22Δ mutant exhibits Etn auxotrophy under PSD1-depressed conditions. Deletion of VID22 in wild-type and PSD1-depressed cells caused partial defects in PE formation through decarboxylation of PS. The enzyme activity of PS decarboxylase in an extract of vid22Δ cells was ∼70% of that in wild-type cells and similar to that in psd2Δ cells and the PS decarboxylase activity remaining in the PSD1-depressed cells became almost negligible with deletion of VID22. Thus, the vid22Δ mutation was suggested to cause a defect in the Psd2p activity. Furthermore, vid22Δ cells were shown to be defective in expression of the PSD2 gene tagged with 6×HA, the defect being ameliorated by replacement of the native promoter of the PSD2 gene with a CYC1 promoter. In addition, an α-galactosidase reporter assay revealed that the activity of the promoter of the PSD2 gene in vid22Δ cells was ∼5% of that in wild-type cells. These results showed that VID22 is required for transcriptional activation of the PSD2 gene.

Phospholipid methylation controls Atg32-mediated mitophagy and Atg8 recycling.

Degradation of mitochondria via selective autophagy, termed mitophagy, contributes to mitochondrial quality and quantity control whose defects have been implicated in oxidative phosphorylation deficiency, aberrant cell differentiation, and neurodegeneration. How mitophagy is regulated in response to cellular physiology remains obscure. Here, we show that mitophagy in yeast is linked to the phospholipid biosynthesis pathway for conversion of phosphatidylethanolamine to phosphatidylcholine by the two methyltransferases Cho2 and Opi3. Under mitophagy-inducing conditions, cells lacking Opi3 exhibit retardation of Cho2 repression that causes an anomalous increase in glutathione levels, leading to suppression of Atg32, a mitochondria-anchored protein essential for mitophagy. In addition, loss of Opi3 results in accumulation of phosphatidylmonomethylethanolamine (PMME) and, surprisingly, generation of Atg8-PMME, a mitophagy-incompetent lipid conjugate of the autophagy-related ubiquitin-like modifier. Amelioration of Atg32 expression and attenuation of Atg8-PMME conjugation markedly rescue mitophagy in opi3-null cells. We propose that proper regulation of phospholipid methylation is crucial for Atg32-mediated mitophagy.

COX assembly factor ccdc56 regulates mitochondrial morphology by affecting mitochondrial recruitment of Drp1.

Mitochondria are dynamic organelles that alter their morphology in response to cellular signaling and differentiation through balanced fusion and fission. In this study, we found that the mitochondrial inner membrane ATPase ATAD3A interacted with ccdc56/MITRAC12/COA3, a subunit of the cytochrome oxidase (COX)-assembly complex. Overproduction of ccdc56 in HeLa cells resulted in fragmented mitochondrial morphology, while mitochondria were highly elongated in ccdc56-repressed cells by the defective recruitment of the fission factor Drp1. We also found that mild and chronic inhibition of COX led to mitochondrial elongation, as seen in ccdc56-repressed cells. These results indicate that ccdc56 positively regulates mitochondrial fission via regulation of COX activity and the mitochondrial recruitment of Drp1, and thus, suggest a novel relationship between COX assembly and mitochondrial morphology.

Involvement of Sac1 phosphoinositide phosphatase in the metabolism of phosphatidylserine in the yeast Saccharomyces cerevisiae.

Sac1 is a phosphoinositide phosphatase that preferentially dephosphorylates phosphatidylinositol 4-phosphate. Mutation of SAC1 causes not only the accumulation of phosphoinositides but also reduction of the phosphatidylserine (PS) level in the yeast Saccharomyces cerevisiae. In this study, we characterized the mechanism underlying the PS reduction in SAC1-deleted cells. Incorporation of (32) P into PS was significantly delayed in sac1∆ cells. Such a delay was also observed in SAC1- and PS decarboxylase gene-deleted cells, suggesting that the reduction in the PS level is caused by a reduction in the rate of biosynthesis of PS. A reduction in the PS level was also observed with repression of STT4 encoding phosphatidylinositol 4-kinase or deletion of VPS34 encoding phophatidylinositol 3-kinase. However, the combination of mutations of SAC1 and STT4 or VPS34 did not restore the reduced PS level, suggesting that both the synthesis and degradation of phosphoinositides are important for maintenance of the PS level. Finally, we observed an abnormal PS distribution in sac1∆ cells when a specific probe for PS was expressed. Collectively, these results suggested that Sac1 is involved in the maintenance of a normal rate of biosynthesis and distribution of PS.

Tam41 is a CDP-diacylglycerol synthase required for cardiolipin biosynthesis in mitochondria.

CDP-diacylglycerol (CDP-DAG) is central to the phospholipid biosynthesis pathways in cells. A prevailing view is that only one CDP-DAG synthase named Cds1 is present in both the endoplasmic reticulum (ER) and mitochondrial inner membrane (IM) and mediates generation of CDP-DAG from phosphatidic acid (PA) and CTP. However, we demonstrate here by using yeast Saccharomyces cerevisiae as a model organism that Cds1 resides in the ER but not in mitochondria, and that Tam41, a highly conserved mitochondrial maintenance protein, directly catalyzes the formation of CDP-DAG from PA in the mitochondrial IM. We also find that inositol depletion by overexpressing an arrestin-related protein Art5 partially restores the defects of cell growth and CL synthesis in the absence of Tam41. The present findings unveil the missing step of the cardiolipin synthesis pathway in mitochondria as well as the flexibile regulation of phospholipid biosynthesis to respond to compromised CDP-DAG synthesis in mitochondria.

Involvement of complex sphingolipids and phosphatidylserine in endosomal trafficking in yeast Saccharomyces cerevisiae.

Sphingolipids play critical roles in many physiologically important events in the yeast Saccharomyces cerevisiae. In this study, we found that csg2Δ mutant cells defective in the synthesis of mannosylinositol phosphorylceramide exhibited abnormal intracellular accumulation of an exocytic v-SNARE, Snc1, under phosphatidylserine synthase gene (PSS1)-repressive conditions, although in wild-type cells, Snc1 was known to cycle between plasma membranes and the late Golgi via post-Golgi endosomes. The mislocalized Snc1 was co-localized with an endocytic marker dye, FM4-64, upon labelling for a short time. The abnormal distribution of Snc1 was suppressed by deletion of GYP2 encoding a GTPase-activating protein that negatively regulates endosomal vesicular trafficking, or expression of GTP-restricted form of Ypt32 GTPase. Furthermore, an endocytosis-deficient mutant of Snc1 was localized to plasma membranes in PSS1-repressed csg2Δ mutant cells as well as wild-type cells. Thus, the PSS1-repressed csg2Δ mutant cells were indicated to be defective in the trafficking of Snc1 from post-Golgi endosomes to the late Golgi. In contrast, the vesicular trafficking pathways via pre-vacuolar endosomes in the PSS1-repressed csg2Δ mutant cells seemed to be normal. These results suggested that specific complex sphingolipids and phosphatidylserine are co-ordinately involved in specific vesicular trafficking pathway.

Impaired retrograde membrane traffic through endosomes in a mutant CHO cell defective in phosphatidylserine synthesis.

Phosphatidylserine (PS), a relatively minor constituent in the plasma membrane (PM), participates in various cellular processes such as clearance of apoptotic cells and recruitment of signaling molecules. PS also localizes in the membranes of endocytic organelles, such as recycling endosomes (REs). We recently showed that in REs, PS binds to the pleckstrin homology (PH) domain of evectin-2, thereby regulating retrograde traffic from REs to the Golgi. However, direct evidence that PS has a role in retrograde traffic is lacking. Here, we examined the contribution of PS to endosomal membrane traffic by exploiting a mutant CHO cell line (PSA-3) that is defective in PS synthesis. In PSA-3 cells, the Golgi localization of TGN38, a protein that circulates between the Golgi and the PM through endosomes by retrograde traffic, was abolished, whereas the localizations of other organelle markers remained unchanged. Increasing the cellular PS level by adding ethanolamine to the culture medium restored the Golgi localization of TGN38. Tracking the endocytic fate of cell surface TGN38 that was labeled by anti-TGN38 antibody showed that retrograde transport of TGN38 was impaired at endosomes, not at the PM. These findings provide direct evidence that intracellular PS is required for retrograde traffic through endosomes.

Hydroxylation state of fatty acid and long-chain base moieties of sphingolipid determine the sensitivity to growth inhibition due to AUR1 repression in Saccharomyces cerevisiae.

The structures of ceramide found in the yeast Saccharomyces cerevisiae are classified into five groups according to the hydroxylation states of the long-chain base and fatty acid moieties. This diversity is created through the action of enzymes encoded by SUR2, SCS7, and as yet unidentified hydroxylation enzyme(s). Aur1p is an enzyme catalyzing the formation of inositol phosphorylceramide in the yeast, and the defect leads to strong growth inhibition due to accumulation of ceramide and reductions in complex sphingolipid levels. In this study, we found that the deletion of SCS7 results in the enhancement of growth inhibition due to repression of AUR1 expression under the control of a tetracycline-regulatable promoter, whereas the deletion of SUR2 attenuates the growth inhibition. Under AUR1-repressive conditions, SCS7 and SUR2 mutants showed reductions in the complex sphingolipid levels and the accumulation of ceramide, like wild-type cells. On the other hand, the deletion of SCS7 had no effect on the growth inhibition through reductions in the complex sphingolipid levels caused by repression of LIP1 encoding a ceramide synthase subunit. Furthermore, the deletion of SUR2 did not suppress the growth inhibition under LIP1-repressive conditions. Therefore, it is suggested that the deletion of sphingolipid hydroxylases changes the toxicity of ceramide under AUR1-repressive conditions.

FMP30 is required for the maintenance of a normal cardiolipin level and mitochondrial morphology in the absence of mitochondrial phosphatidylethanolamine synthesis.

Mitochondria of the yeast Saccharomyces cerevisiae contain enzymes Crd1p and Psd1p, which synthesize cardiolipin (CL) and phosphatidylethanolamine respectively. A previous study indicated that crd1Δ is synthetically lethal with psd1Δ. In this study, to identify novel genes involved in CL metabolism, we searched for genes that genetically interact with Psd1p, and found that deletion of FMP30 encoding a mitochondrial inner membrane protein results in a synthetic growth defect with psd1Δ. Although fmp30Δ cells grew normally and exhibited a slightly decreased CL level, fmp30Δpsd1Δ cells exhibited a severe growth defect and an about 20-fold reduction in the CL level, as compared with the wild-type control. We found also that deletion of FMP30 caused a defect in mitochondrial morphology. Furthermore, FMP30 genetically interacted with seven mitochondrial morphology genes. These results indicated that Fmp30p is involved in the maintenance of mitochondrial morphology and required for the accumulation of a normal level of CL in the absence of mitochondrial phosphatidylethanolamine synthesis.

Requirement of a specific group of sphingolipid-metabolizing enzyme for growth of yeast Saccharomyces cerevisiae under impaired metabolism of glycerophospholipids.

Sphingolipids play critical roles in many physiologically important events in yeast Saccharomyces cerevisiae. In this study, we screened for yeast mutants showing high sensitivity to Aureobasidin A, an inhibitor of inositol phosphorylceramide synthase, and found that a lack of SAC1 encoding phosphoinositides phosphatase causes high sensitivity to the inhibitor. Double mutation analysis involving the SAC1 and non-essential sphingolipid-metabolizing enzyme genes revealed that csg1Δ, csg2Δ, ipt1Δ or scs7Δ causes synthetic lethality with deletion of SAC1. As previously reported, SAC1-repressed cells exhibited a reduced cellular phosphatidylserine (PS) level, and overexpression of PSS1 encoding PS synthase complemented the growth defects of scs7Δ, csg1Δ and ipt1Δ cells under SAC1-repressive conditions. Furthermore, repression of PSS1 expression resulted in synthetic growth defect with the deletion of CSG1, IPT1 or SCS7. The growth defects of scs7Δ, csg1Δ and ipt1Δ cells under SAC1- or PSS1-repressive conditions were also complemented by overexpression of Arf-GAP AGE1, which encodes a protein related to membrane trafficking. Under SAC1-repressive conditions, scs7Δ, csg1Δ and ipt1Δ cells showed defects in vacuolar morphology, which were complemented by overexpression of each of PSS1 and AGE1. These results suggested that a specific group of sphingolipid-metabolizing enzyme is required for yeast cell growth under impaired metabolism of glycerophospholipids.

Defect of synthesis of very long-chain fatty acids confers resistance to growth inhibition by inositol phosphorylceramide synthase repression in yeast Saccharomyces cerevisiae.

Aureobasidin A (AbA) inhibits Aur1p, an enzyme catalysing the formation of inositol phosphorylceramide in the yeast Saccharomyces cerevisiae. AbA treatment results not only in reductions in complex sphingolipid levels but also in accumulation of ceramides, both of which are believed to lead to the growth defect caused by this inhibitor. We screened for mutants showing resistance to this drug, and found that a lack of ELO3, the gene involved in synthesis of very long-chain fatty acids, confers resistance to the inhibitor. The resistance as to growth inhibition by reduction in Aur1p activity was also confirmed by repression of AUR1 expression under the control of a tetracycline-regulatable promoter. Under the AUR1-repressive conditions, the ELO3 mutant showed reduction in the complex sphingolipid levels and the accumulation of ceramide, like wild-type cells. However, with repression of LCB1 encoding serine palmitoyltransferase or LIP1 encoding the ceramide synthase subunit, the ELO3 mutation did not confer resistance to growth inhibition induced by the impaired sphingolipid biosynthesis. Therefore, it is suggested that the ELO3 mutant shows resistance as to accumulation of ceramides, implying that the chain lengths of fatty acids in ceramide are a critical factor for the ceramide-induced growth defect under AUR1-repressive conditions.

The AAA+ ATPase ATAD3A controls mitochondrial dynamics at the interface of the inner and outer membranes.

Dynamic interactions between components of the outer (OM) and inner (IM) membranes control a number of critical mitochondrial functions such as channeling of metabolites and coordinated fission and fusion. We identify here the mitochondrial AAA(+) ATPase protein ATAD3A specific to multicellular eukaryotes as a participant in these interactions. The N-terminal domain interacts with the OM. A central transmembrane segment (TMS) anchors the protein in the IM and positions the C-terminal AAA(+) ATPase domain in the matrix. Invalidation studies in Drosophila and in a human steroidogenic cell line showed that ATAD3A is required for normal cell growth and cholesterol channeling at contact sites. Using dominant-negative mutants, including a defective ATP-binding mutant and a truncated 50-amino-acid N-terminus mutant, we showed that ATAD3A regulates dynamic interactions between the mitochondrial OM and IM sensed by the cell fission machinery. The capacity of ATAD3A to impact essential mitochondrial functions and organization suggests that it possesses unique properties in regulating mitochondrial dynamics and cellular functions in multicellular organisms.