Amino acid residues important for substrate specificity of the amino acid permeases Can I p and Gnp I p in Saccharomyces cerevisiae

Deletion of the general amino acid permease gene GAP1 abolishes uptake of L -citrulline in Saccharomyces cerevisiae, resulting in the inability to grow on L-citrulline as sole nitrogen source. Selection for suppressor mutants that restored growth on L-citrulline led to isolation of 21 mutations in th e arginine permease gene CAN1. One similar mutation was found in the glutam ine-asparagine permease gene GNP1. L-[C-14]citrulline uptake measurements c onfirmed that suppressor mutations in CAN1 conferred uptake of this amino a cid, while none of the mutant permeases had lost the ability to transport L -[C-14]arginine. Substrate specificity seemed to remain narrow in most case s, and broad substrate specificity was only observed in the cases where mut ations affect two proline residues (P148 and P313) that are both conserved in the amino acid-polyamine-choline (APC) transporter superfamily. We found mutations affecting six predicted domains (helices III and X, and loops 1. 2, 6 and 7) of the permeases. Helix III and loop 7 are candidates for doma ins in direct contact with the transported amino acid. Helix III was affect ed in both CAN1 (Y173H, Y173D) and GNP1 (W239C) mutants and has previously been found to be important for substrate preference in other members of the family. Furthermore, the mutations affecting loop 7 (residue T354, S355, Y 356) are close to a glutamate side chain (E367) potentially interacting wit h the positively charged substrate, a notion supported by conservation of t he side chain in permeases for cationic substrates. Copyright (C) 2001 John Wiley & Sons, Ltd.