The Alanine, Serine, Cysteine Transporters (ASCTs) mediate the Na+-dependent transport of neutral amino acids. ASCT2 is the predominant glutamine transport system for human cancer cells, and is upregulated across various types of cancer. ASCT1 and ASCT2 share 57% amino acid sequence identity, however they display varying substrate selectivity profiles. ASCT1 transports small neutral amino acids including alanine, serine and cysteine. ASCT2 transports a broader range of substrates, including glutamine, asparagine and methionine. In this study, we investigate the molecular basis for differences in substrate selectivity between the ASCTs. Mutating the key substrate binding residue in ASCT1 to the ASCT2 equivalent (T459C) introduced glutamine sensitivity. The additional mutation T458S to create T458S/T459C increased the glutamine affinity to reflect that of ASCT2. Similarly, glutamyl-p-nitroanilide (GPNA) is an inhibitor of ASCT2 but not ASCT1. T459C did not display an increased sensitivity to GPNA, however sensitivity was increased in the double mutant T458S/T459C. The reverse mutations in ASCT2 (S481T, C482T) diminished glutamine binding, and also reduced the sensitivity of ASCT2 to GPNA.
The current structural model for ASCTs is the homologous prokaryotic aspartate transporter GltPh. Crystal structures of GltPh have provided a wealth of information regarding the transport mechanisms of ASCTs, however information specific to neutral amino acid transport is lacking. The mutation R397C in GltPh diminished acidic amino acid transport and introduced serine and alanine transport, as well as an increased sensitivity to a wide range of neutral amino acids. This provides a useful model for neutral amino acid transport.