Amino acid residues important for substrate specificity of the amino acid permeases Can1p and Gnp1p in Saccharomyces cerevisiae

Abstract

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 the arginine permease gene CAN1. One similar mutation was found in the glutamine-asparagine permease gene GNP1. L-[(14)C]citrulline uptake measurements confirmed that suppressor mutations in CAN1 conferred uptake of this amino acid, while none of the mutant permeases had lost the ability to transport L-[(14)C]arginine. Substrate specificity seemed to remain narrow in most cases, and broad substrate specificity was only observed in the cases where mutations 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 domains in direct contact with thetransported amino acid. Helix III was affected 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, Y356) are close to a glutamate side chain (E367) potentially interacting with the positively charged substrate, a notion supported by conservation of the side chain in permeases for cationic substrates.

Fitness landscape of substrate-adaptive mutations in evolved APC transporters

“…The differential Cit uptake rates of the single and double mutants showcase how in vivo experimentally generated single mutations can alter the substrate spectrum of the yeast amino acid transporters without the loss of ancestral functions. Our observations are in line with studies of substrate promiscuity being the result of adaptive mutations in different membrane transporters [41–47]. The altered specificity for a substrate, which is mirrored in our case in the altered fitness, could be a result of a different affinity constant for the amino acid and/or maximal transport rate.…”

“…Our observations are in line with studies of substrate promiscuity being the result of adaptive mutations in different membrane transporters. [39]–[45] The altered specificity for a substrate, which is mirrored in our case in the altered fitness, could be a result of a different affinity constant for the amino acid and/or maximal transport rate.…”

“…I412 in TM8 is part of the conserved sequence [IV]-L-[ITAVL] and thus in AGP1 -V and AGP1 -NV, Ile is changed to the only other common residue, Val, which is found in DIP5 , LYP1 and ALP1 [53]. Reportedly, single site substitutions in conserved regions of CAN1 and GNP1 have been shown to broaden the substrate range of these YAT transporters and accommodate for the uptake of Cit [55]. Substitutions in non-conserved regions can also have an effect on a transporter’s substrate spectrum.…”

“…Reportedly, single site substitutions in conserved regions of CAN1 and GNP1 have been shown to broaden the substrate range of these YAT transporters and accommodate for the uptake of Cit [55]. Substitutions in non-conserved regions can also have an effect on a transporter's substrate spectrum.…”

Nitrogen catabolite repressible GAP1 promoter, a new tool for efficient recombinant protein production in S. cerevisiae

“…Such quantities of purified Gap1 allow proceeding with in vitro experiments and envisaging structural biology techniques. Although many studies have deciphered the regulation of the protein, both at the transcription and the protein level [13,37,38], little is known about the transport mechanism and the substrate recognition [39,40]. We expect that this production tool will facilitate in vitro studies and overall understanding of the protein.…”