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.Mitochondria not only supply cells with the bulk of their ATP but also contribute to the fine regulation of metabolism, calcium homeostasis, and apoptosis (
27). Coordination of these functions is dependent on the dynamic nature of mitochondria (
5). These organelles constantly fuse and divide to form small spheres, short rods, or long tubules and are actively transported to specific subcellular locations. These processes are essential for mammalian development, and defects can lead to degenerative diseases and cancers (
9,
17). In eukaryotes, these organellar gymnastics are controlled by numerous pathways that preserve proper mitochondrial morphology and function (
30,
45). The best-understood mitochondrial process is the fusion and fission pathways, which rely on conserved GTPases, and their binding partners to regulate organelle connectivity (
10,
18,
45). There are also evidences that dynamic interactions between the outer membrane (OM) and inner membrane (IM) exist for coordinated fusion and fission, channeling of metabolites, and protein transport, but proteins playing a role in these interactions have yet to be identified (
34). In the present study, we provide a detailed biochemical and functional characterization of the mitochondrial AAA
+ ATPase ATAD3A protein that is present exclusively in multicellular eukaryotes and which participates in the control of mitochondrial dynamics at the interface between the IMs and OMs. Proteins related to the
Atad3A genes have been previously identified in proteomic surveys of mouse brain mitochondria (
28) and liver mitochondrial inner membrane (
8), as mitochondrial DNA-binding proteins (
4,
21,
44) and as nuclear mRNA-associated proteins (
6). The
Atad3A protein has also been identified as a cell surface antigen in some human tumors (
16). Functional genomics identified the
Drosophila Atad3A ortholog (
bor) as a major gene positively regulated by the TOR (for target of rapamycin) signaling pathway involved in cell growth and division (
19). In our laboratory, we identified ATAD3A as a specific target for the Ca
2+/Zn
2+-binding S100B protein (B. Gilquin et al., unpublished data). We here show that ATAD3A is anchored into the mitochondrial IM at contact sites with the OM. The N-terminal domain of ATAD3A interacts with the inner surface of the OM and its C-terminal AAA ATPase domain localizes in a specific matrix compartment. Thanks to its simultaneous interaction with two membranes, ATAD3A regulates mitochondrial dynamics at the interface between the IMs and OMs and controls diverse cell responses ranging from cell growth, channeling of cholesterol, and mitochondrial fission.
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