LAUSR

ATP‐binding cassette (ABC) systems, characterized by ABC‐type nucleotide‐binding domains (NBDs), play crucial roles in various aspects of human physiology. Human ABCG5 and ABCG8 form a heterodimeric transporter that functions in the efflux of sterols. We used sequence similarity search, multiple sequence alignment, phylogenetic analysis, and structure comparison to study the evolutionary origin and sequence signatures of ABCG5 and ABCG8. Orthologs of ABCG5 and ABCG8, supported by phylogenetic analysis and signature residues, were identified in bilaterian animals, Filasterea, Fungi, and Amoebozoa. Such a phylogenetic distribution suggests that ABCG5 and ABCG8 could have originated in the last common ancestor of Amorphea (the unikonts), the eukaryotic group including Amoebozoa and Opisthokonta. ABCG5 and ABCG8 were missing in genomes of various lineages such as snakes, jawless vertebrates, non‐vertebrate chordates, echinoderms, and basal metazoan groups. Amino‐acid changes in key positions in ABCG8 Walker A motif and/or ABCG5 C‐loop were observed in most tetrapod organisms, likely resulted in the loss of ATPase activity at one nucleotide‐binding site. ABCG5 and ABCG8 in Ecdysozoa (such as insects) exhibit elevated evolutionary rates and accumulate various changes in their NBD functional motifs. Alignment inspection revealed several residue positions that show different amino‐acid usages in ABCG5/ABCG8 compared to other ABCG subfamily proteins. These residues were mapped to the structural cores of transmembrane domains (TMDs), the NBD‐TMD interface, and the interface between TMDs. They serve as sequence signatures to differentiate ABCG5/ABCG8 from other ABCG subfamily proteins, and some of them may contribute to substrate specificity of the ABCG5/ABCG8 transporter.