Elevator-type transporters are a group of proteins translocating nutrients and metabolites across cell membranes. Despite structural and functional differences, elevator-type transporters use a common mechanism of substrate translocation via reversible movements of a mobile core domain (the elevator), which includes the substrate binding site, along a rigid scaffold domain, stably anchored in the plasma membrane. How substrate specificity is determined in elevator transporters remains elusive. Here, I discuss how a recent report on the sliding elevator mechanism, seen under the context of genetic analysis of a prototype fungal transporter, sheds light on how specificity might be genetically modified. I propose that flexible specificity alterations might occur by 'loosening' of the sliding mechanism from tight coupling to substrate binding.

Members of the ubiquitous Nucleobase Ascorbate Transporter (NAT) family are H or Na symporters specific for the cellular uptake of either purines and pyrimidines or L-ascorbic acid. Despite the fact that several bacterial and fungal members have been extensively characterised at a genetic, biochemical or cellular level, and crystal structures of NAT members from Escherichia coli and Aspergillus nidulans have been determined pointing to a mechanism of transport, we have little insight on how substrate selectivity is determined. Here, we present systematic mutational analyses, rational combination of mutations, and novel genetic screens that reveal cryptic context-dependent roles of partially conserved residues in the so-called NAT signature motif in determining the specificity of the UapA transporter of A. nidulans. We show that specific NAT signature motif substitutions, alone and in combinations with each other or with distant mutations in residues known to affect substrate selectivity, lead to novel UapA versions possessing variable transport capacities and specificities for nucleobases. In particular, we show that a UapA version including the quadruple mutation T405S/F406Y/A407S/Q408E in the NAT signature motif (UapA-SYSE) becomes incapable of purine transport, but gains a novel pyrimidine-related profile, which can be further altered to a more promiscuous purine/pyrimidine profile when combined with replacements at distantly located residues, especially at F528. Our results reveal that UapA specificity is genetically highly modifiable and allow us to speculate on how the elevator-type mechanism of transport might account for this flexibility.