In vivo and in vitro studies of the purine-cytosine permease of Saccharomyces cerevisiae. Functional analysis of a mutant with an altered apparent transport constant of uptake.
The FCY2 gene of the purine-cytosine permease (PCP) of Saccharomyces cerevisiae and the allele fcy2-21 have been cloned on the yeast multicopy plasmid pJDB207. The corresponding plasmids were introduced into a S. cerevisiae strain carrying a chromosomal deletion at the FCY2 locus. The resulting strains were designated pAB4 and pAB25 respectively. The pAB25 strain, which carries the fcy2-21 allele, contains four amino acid changes in the open reading frame of the PCP (Weber et al., 1989). The influence of these mutations was studied on cells by determination of the uptake constants of purine bases and cytosine [apparent Michaelis constant of transport (Ktapp) and Vmax] and on plasma-membrane preparations, by measurements of binding parameters at equilibrium [(Kd and maximum amount of binding sites/Bmax)]. For strain pAB4, the Ktapp and Vmax of uptake were almost similar for all solutes considered [1.8-2.6 microM and 8.5-10.2 nmol.min-1.(10(7) cells)-1]. The main effect of the mutations in strain pAB25 was based on a large increase in Ktapp for all ligands except adenine. Plasma membranes of each strain displayed one class of specific binding sites. Variations in Kd of 0.4-1 microM were observed for pAB4. These slight variations had no effect on the Ktapp of uptake measured for the corresponding solutes. In contrast, using pAB25 membranes, Kd increased dramatically; 2.6 microM, 40 microM and 96 microM for adenine, cytosine and hypoxanthine, respectively. These increments were correlated to variations in Ktapp of the uptake for cytosine and hypoxanthine. Therefore, we conclude that modification in the Ktapp of uptake in the strain carrying fcy2-21 allele is merely due to a modification of the binding ability of the permease for its ligands.
Brèthes
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D
Institut de Biochimie Cellulaire et de Neurochimie, Centre National de la Recherche Scientifique, Bordeaux, France.
Chirio
M C
MC
Napias
C
C
Chevallier
M R
MR
Lavie
J L
JL
Chevallier
J
J
D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D015825:Chromosomes, Fungal; D003596:Cytosine / Q000378:metabolism; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D033702:Nucleobase Transport Proteins; D010957:Plasmids; D011687:Purines / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins
D002352:Carrier Proteins; C043181:FCY2 protein, S cerevisiae; D026901:Membrane Transport Proteins; D033702:Nucleobase Transport Proteins; D011687:Purines; D029701:Saccharomyces cerevisiae Proteins; D003596:Cytosine; C030985:purine