Evolution of Substrate Selectivity in ATP Binding Cassette Exporters

Abstract

ATP binding cassette proteins represent the largest super-family of primary transporters with homologous sequences present in all extant organisms, coordinating the hydrolysis of ATP in nucleotide binding domains (NBDs) with the transport of substrates across biological membranes through transmembrane domains (TMDs). Type I ABC exporters are a large sub-family of ABC proteins that play a role in the unidirectional transport of substrates from the cytosol to the extracellular space or organelle lumens. Paralogous exporters have evolved to transport a wide variety of substrates across evolution like LPS in gram-negative bacteria, intermediates for cytosolic Fe-S cluster synthesis in eukaryotes and antigenic peptides in adaptive immunity of Vertebrates. The evolutionary flexibility of substrate selectivity in type I ABC exporters is particularly interesting given the structural and mechanistic similarity across paralogs. My doctoral work took advantage of a type I exporter that plays the role of a dedicated pheromone exporter in fungal mating to address the open question of substrate selectivity in exporters. Fungal mating has pairs of lipidated peptide pheromones and dedicated ABC exporters conserved across 500 million years of evolution. The sequences of peptide pheromones change across the fungal lineage leading to co-evolution of exporters with their cognate pheromone substrates. The ability to grow dense populations rapidly and powerful genetics make the baker’s yeast, S. cerevisiae an ideal model organism to build experimental systems that test functional variation in proteins. We built an experimental system that quantitatively couples the pheromone export activity of an expressed ABC exporter to a fluorescent reporter in the same cell. Starting from a mutated set of a non-functional orthologous exporter, we selected for increased pheromone export based on fluorescence of the cells. Our work shows that there is a large target size for mutations that affect substrate selectivity of ABC exporters. A large target size with additive contributions of individual mutations might explain the evolutionary success of ABC exporters. Specifically, a large target size would provide high probability trajectories for duplication and divergence of a transporter to transport “novel” substrates. Increasing availability of sequenced genomes of fungi also provides a library of substrates (pheromones) and exporters to test the model for co-evolution. Orthologous pheromone exporters can be identified by homology search, while pheromones candidates can be identified by an algorithmic sieve that we have developed. Going forward, our experimental selection system can enable comparisons of phylogenetic variation in pheromone exporters we observe by imposing artificial selection.