Abstract
SUMMARYATP-binding cassette (ABC) transporters are the largest family of ATP-hydrolyzing transporters, which import or export substrates across membranes, and have members in every sequenced genome. Structural studies and biochemistry highlight the contrast between the global structural similarity of homologous transporters and the enormous diversity of their substrates. How do ABC transporters evolve to carry such diverse molecules and what variations in their amino acid sequence alter their substrate selectivity? We mutagenized the transmembrane domains of a conserved fungal ABC transporter that exports a mating pheromone and selected for mutants that export a non-cognate pheromone. Mutations that alter export selectivity cover a region that is larger than expected for a localized substrate-binding site. Individual selected clones have multiple mutations, which have broadly additive contributions to specific transport activity. Our results suggest that multiple positions influence substrate selectivity, leading to alternative evolutionary paths toward selectivity for particular substrates and explaining the number and diversity of ABC transporters.
Highlights
During evolution, many genes have duplicated and diverged to acquire new functions
The efficiency of mating roughly correlates with the phylogenetic distance between S. cerevisiae and the yeast containing the supplied Ste6 ortholog, which is consistent with coevolution of the transporter with the pheromone it transports (Figure 1B)
To confirm that the efficiency of mating is a good measure of transporter function, we assayed pheromone export directly by collecting exported a-factor from growing cultures and using a serial dilution bioassay to measure the quantity of exported pheromone (Figure 1C)
Summary
Many genes have duplicated and diverged to acquire new functions. ATP-binding cassette (ABC) transporters, the largest single family of proteins in the Protein Families (PFAM) database, are an excellent example of protein diversification. ABC transporters contain cytoplasmic nucleotide-binding domains (NBDs) with conserved motifs for binding and hydrolyzing ATP, connected to transmembrane domains (TMDs) that undergo conformational changes to transport substrates across membranes [1, 2]. Sequence conservation in the TMDs of homologous exporters has not been helpful in identifying a conserved binding site for cognate substrates in orthologous or paralogous exporters. ABC transporters oscillate between two states: one with a central substrate-binding cavity exposed to the cytoplasm (inward-open) and one with roughly the same surface exposed to the other side of the membrane (outward-open) [2]. Biochemical crosslinking and mutagenesis experiments on P-gp and TAP have identified positions in the TMD cavity involved in substrate recognition [8,9,10], but a system to characterize the sequence determinants of substrate selectivity in type I ABC exporters has not been reported
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