TY - JOUR

T1 - Amino acid residues important for substrate specificity of the amino acid permeases Can l p and Gnp l p in Saccharomyces cerevisiae

AU - Regenberg, Birgitte

AU - Kielland-Brandt, Morten C.

PY - 2001/12/5

Y1 - 2001/12/5

N2 - 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-[14C]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-[14C]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 the transported 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.

AB - 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-[14C]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-[14C]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 the transported 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.

KW - Arginine transport

KW - CANI

KW - Citrulline

KW - Glutamine transport

KW - GNPI

KW - YDR508c

KW - YEL063c

UR - http://www.scopus.com/inward/record.url?scp=0035182926&partnerID=8YFLogxK

U2 - 10.1002/yea.792

DO - 10.1002/yea.792

M3 - Journal article

C2 - 11746604

AN - SCOPUS:0035182926

VL - 18

SP - 1429

EP - 1440

JO - Yeast

JF - Yeast

SN - 0749-503X

IS - 15

ER -