Upon discovery of the first archaeal species in the 1970s, life has been subdivided into three domains: Eukarya, Archaea, and Bacteria. However, the organization of the three-domain tree of life has been challenged following the discovery of archaeal lineages such as the TACK Superphylum and, more recently, the Asgard Superphylum. The Asgard superphylum has emerged as the closest archaeal ancestor to eukaryotes and may allow us to improve our understanding of the evolution of life from relatively simple prokaryotes to complex eukaryotes. In this study, we characterize the transportomes and their substrates within four metagenomes of Asgard archaea (i.e., Loki-, Odin-, Thor-, and Heimdall-archaeota). Using the Transporter Classification Database (TCDB) as reference, candidate transporters were identified based on sequence similarity, alignment coverage, overlap of hydropathy profiles, TMS topologies and shared Pfam domain content. Identified transport systems are compared within the Asgard superphylum, and to other eukaryotic, archaeal and bacterial transport systems. We found that Asgard organisms rely mostly on secondary carriers for the transport of cofactors and vitamins. They seem to contain a diverse range of systems required for establishing the membrane potential and proton motive force for subsequent ATP production. To varying degrees, the results indicate that Asgard organisms depend mostly on the intake of organic molecules such as lipids, amino acids, and proteins. The transporters identified clearly resemble prokaryotic transporter more than eukaryotic specific transporters. Taken together, the results confirm the mixotrophic and diverse metabolic capabilities of the Asgard superphylum.

The Transporter Classification Database (TCDB) provides literature-curated, functional and evolutionary information on transporters, as well as descriptions of their families and superfamilies across all domains of life. This project aims to test the hypothesis that the Ligand-gated Ion Channel (LIC) family is a member of the Voltage-gated Ion Channels (VIC) superfamily. The topological similarities between the Glutamate-gated Ion Channel (GIC) family, an established member of the VIC superfamily, and the LIC family provided the initial clue of the relationship. To test our hypothesis, multiple bioinformatic approaches were applied to explore the similarities between the LIC family and existing members of the VIC superfamily. These included (1) sequence similarity, (2) compatibility of topology and hydropathy profiles, (3) shared domains, (4) conserved motifs, and (5) similarity of sequence profiles at the family level. Our results showed that two families from the VIC superfamily, the Glutamate-gated Ion Channel (GIC) Family of Neurotransmitter Receptors and the Voltage-gated Proton Channel (VPC) Family, generated reliable signals and satisfied at least five of our six criteria, thus supporting the conclusion that the LIC family is a distant member of the VIC superfamily.

ATP-binding Cassette (ABC) transporters use ATP as an energy source and move a variety of substrates concentratively across cellular membranes. Previous studies based on primary protein sequence data suggested that integral membrane ABC exporters evolved independently at least three times, giving rise to three ABC types. Given the increasing availability of ABC structures in the Protein Data Bank (PDB) and the substantially larger number of primary sequences, we could investigate whether the current data support the conclusions obtained based on sequence analyses alone. We conducted sequence and structural analyses on the transmembrane domains (TMDs) and the nucleotide binding (ATPase) domains (NBDs) of the three proposed ABC types, ABC1 in which a repeat unit of 2 TMSs triplicated, ABC2 in which a repeat unit of 3 TMSs duplicated, and ABC3 in which a repeat unit of 4 TMSs duplicated. The three most divergent families of the 70 ABC exporter families were excluded from our studies. The clustering patterns of both the TMDs and the NBDs showed that ABC1 forms a monophyletic group, whereas ABC2 and ABC3 share a major branch. The topological similarities of the two trees for the TMDs and NBDs strongly support the notion that these two domains have co-evolved. Based on sequence and structural divergence as well as organismal distribution, we suggest that ABC2s evolved first, followed by ABC1, and then ABC3. Our results provide insight into the evolutionary relationships between ABC types and serve as a guide for future studies of the ABC superfamily.

Determining the relatedness between biological entities can translate to knowledge which would otherwise be difficult to uncover without greater context. In particular, identifying homology between proteins often provides insight on previous unknowns such as structure, function, and even substrate identity. Given that transport proteins can be found not just in cells, but also in viruses, the ability to relate viroporin protein families with their eukaryotic and bacterial counterparts is an exciting development in superfamily formation. Using a number of bioinformatic techniques to compare the Ephemerovirus Viroporin (EVVP) family, the Rhabdoviridae Putative Viroporin (RV-U5) family, the Phospholemman (PLM) family, and the Small Integral Membrane Protein (SIMP) family, we examined the protein families as candidates for a superfamily on the basis of protein sequence similarity, compatibility of hydropathy profiles, topology of transmembrane segments (TMSs) and conservation of protein domains and sequence motifs. Our results indicate that the Pfam domains ATP1G1_PLM_MAT8 (PF02038) and DUF4713 (PF15831) can be found in or projected onto all four families. In addition, we identified a 26-residue motif conserved across the superfamily which is located within the PF02038 domain. This motif is characterized by hydrophobic residues that help anchor the protein to the membrane and charged residues that constitute phosphorylation sites. Taken together, these pieces of evidence justify the creation of the novel Phospholemman/SIMP/Viroporin (PSV) Superfamily.