Amino acid residues important for substrate specificity of the amino acid permeases Can1p and Gnp1p 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 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.
Regenberg
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Department of Physiology, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark.
Kielland-Brandt
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MC
D000595:Amino Acid Sequence; D026905:Amino Acid Transport Systems / Q000235:genetics / Q000378:metabolism*; D026922:Amino Acid Transport Systems, Acidic / Q000235:genetics / Q000378:metabolism*; D002956:Citrulline / Q000378:metabolism; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D026905:Amino Acid Transport Systems; D026922:Amino Acid Transport Systems, Acidic; C097640:CAN1 protein, Candida albicans; D004271:DNA, Fungal; D005656:Fungal Proteins; C429263:GNP1 protein, S cerevisiae; D026901:Membrane Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; D002956:Citrulline