ATP transporter types.

(a) Nucleotide transporter (NTT) present in intracellular bacterial parasite that imports ATP from host cell cytoplasm. Parasite NTT features all alpha 12 TM containing MFS general substrate transporter fold (SCOP id: 2000248). (b) ATP/ADP translocator 1 (NTT1) present in plastid inner membrane responsible for importing ATP from plant cell cytoplasm. Plastid NTTs feature all alpha 12 TM containing MFS general substrate transporter fold (SCOP id: 2000248). (c) Mitochondrial ATP/ADP translocator (ADT/ANT/AAC) that resides in mitochondrial and mitochondria derived organelles that exports ATP to the eukaryotic cell cytoplasm. AACs feature all alpha 6 TM bundle Mitochondrial carrier fold (SCOP id: 2000266).

Mitochondrial ATP-ADP carriers (AACs) are the pioneer members of SLC25 family.

(a) Tree showing all the Eukaryotic supergroups and the phylogenetic position of A. godoyi, S. cerevisiae and P. tetraurelia in three evolutionarily distinct eukaryotic supergroups that have diverged at the stage of LECA. (b) Maximum likelihood phylogenetic tree of SLC25 carrier family members from A. godoyi, S. cerevisiae and P. tetraurelia. Branch support of bootstrap value ≥ 90 are indicated by black circles.

Structure guided screen of AACs in AlphaFold structure database of prokaryote proteome.

(a) X-ray crystal structure of Saccharomyces cerevisiae AAC and its secondary structure topology. (b) AlphaFold predicted structure model of Andalucia godoyi AAC. (c) Schematic diagram of the pipeline employed in this study leveraging protein 3D-structrure search for identification of potential remote homologue of AACs in archaea and bacteria. (d) DALI Z-score, (e) TM-score, (f) sequence identity of archaeal and bacterial structure search hits of AACs.

Putative homologue of AAC in prokaryotes.

(a) Schematic flowchart depicting the approach for selection of the candidate homologue of AACs in prokaryote structural hits based on presence or absence of the structural hits in the bacterial and archaeal phyla. (b) Gene conservation of bacterial structural search hits of AAC across bacterial phyla. (c) Gene conservation of archaeal hits of structural search of AAC in archaeal kingdoms and phyla. (d) AlphaFold predicted model of E. coli YihY and its predicted transmembrane topology. (e) AlphaFold predicted model of E. coli CysZ and its predicted transmembrane topology.

AAC is structurally related to prokaryotic proteins through circular permutation of a TM-helix.

(a) Secondary structure topology and six TM-helix of AAC, YihY and CysZ (lateral and axial views). (b) Primary structure of AAC, YihY and CysZ showing circular permutation of one TM-helix. (c) Hypothetical circular permuted YihYCP and CysZCP featuring similar secondary structure topology as to that of AAC. (d) Axial view of the six TM-helix topology of YihYCP and CysZCP. Secondary structure topology of (e) YihYCP and (f) CysZCP. (g) AlphaFold predicted structure model of YihYCP and its superimposition on YihY and AAC. (h) AlphaFold predicted structure model of CysZCP and its superimposition on CysZ and AAC. (i) DALI Z-score and (j) TM-score for structural superimposition of AAC on AlphaFold predicted structure model of circular permuted bacterial and archaeal structure search hits.

Conservation of MCF motif in CysZ.

(a) Secondary structure topology of AAC and the three-fold internal symmetry of two TM-helix containing repeat. (b) Topology of CysZ and its two TM-helix repeats. Structure of (c) AAC repeat 1, (d) AAC repeat 2 (e) AAC repeat 3, (f) CysZ repeat 2, (g) CysZ repeat 3. (h) Structural overlay of AAC repeat 2 and CysZ repeat 2. (i) Structural overlay of AAC repeat 3 and CysZ repeat 3. (j) MCF motif in AAC repeat 1, 2 and 3. (k) MCF motif in CysZ repeat 3.

Model for evolution of mitochondrial AAC from bacterial CysZ.

Schematic model depicting proposed origin of mitochondrial ATP exporter (AAC) from the bacterial sulfate transporter CysZ during the endosymbiotic origin of mitochondria in eukaryotic cells from bacterial endosymbiont. The bacterial sulfate transporter CysZ, involved in the initial metabolic syntrophy that spurred the formation of proto-mitochondria was eventually evolved and re-purposed for ATP export in mitochondria at the stage of LECA. Evolution of AAC from bacterial sulfate transporter CysZ is a foundational step of mitochondrial emergence, and therefore the onset of eukaryotic cell complexities.