Characterization of the transport mechanism and permeant binding profile of the uridine permease Fui1p of Saccharomyces cerevisiae.
The uptake of Urd into the yeast Saccharomyces cerevisiae is mediated by Fui1p, a Urd-specific nucleoside transporter encoded by the FUI1 gene and a member of the yeast Fur permease family, which also includes the uracil, allantoin, and thiamine permeases. When Fui1p was produced in a double-permease knock-out strain (fur4Deltafui1Delta) of yeast, Urd uptake was stimulated at acidic pH and sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone. Electrophysiological analysis of recombinant Fui1p produced in Xenopus oocytes demonstrated that Fui1p-mediated Urd uptake was dependent on proton cotransport with a 1:1 stoichiometry. Mutagenesis analysis of three charged amino acids (Glu(259), Lys(288), and Asp(474) in putative transmembrane segments 3, 4, and 7, respectively) revealed that only Lys(288) was required for maintaining high Urd transport efficiency. Analysis of binding energies between Fui1p and different Urd analogs indicated that Fuip1 interacted with C(3')-OH, C(2')-OH, C(5)-H, and N(3)-H of Urd. Fui1p-mediated transport of Urd was inhibited by analogs with modifications at C-5', but was not inhibited significantly by analogs with modifications at C-3', C-5, and N-3 or inversions of configuration at C-2' and C-3'. This characterization of Fui1p contributes to the emerging knowledge of the structure and function of the Fur family of permeases, including the Fui1p orthologs of pathogenic fungi.
Zhang
Jing
J
Membrane Protein Research Group and the Department of Oncology, University of Alberta, Alberta T6H 1Z2, Canada.
Smith
Kyla M
KM
Tackaberry
Tracey
T
Sun
Xuejun
X
Carpenter
Pat
P
Slugoski
Melissa D
MD
Robins
Morris J
MJ
Nielsen
Lars P C
LP
Nowak
Ireneusz
I
Baldwin
Stephen A
SA
Young
James D
JD
Cass
Carol E
CE
D000818:Animals; D006863:Hydrogen-Ion Concentration; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D033703:Nucleoside Transport Proteins; D021381:Protein Transport; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014529:Uridine / Q000378:metabolism; D014981:Xenopus
C413444:FUI1 protein, S cerevisiae; D026901:Membrane Transport Proteins; D033703:Nucleoside Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; D014529:Uridine