In vivo phosphorylation of the yeast uracil permease.
The uptake of uracil by the yeast Saccharomyces cerevisiae is mediated by a specific permease encoded by the FUR4 gene. This uracil permease is a multispanning membrane protein that follows the secretory pathway to the plasma membrane. We have used in vivo pulse labeling and immunoprecipitation to show that the uracil permease is phosphorylated. Phosphoamino acid analysis indicates that the phosphorylation occurs on seryl residues. Experiments with temperature sensitive secretory mutants, blocked at successive steps of the secretory pathway, have established that the phosphorylation of the permease takes place at the plasma membrane. Under steady state conditions, Western immunoblotting showed multiple phosphorylated permease species. Their relative abundance appeared susceptible to metabolic conditions. This study is, therefore, a first step toward identifying a molecular mechanism involved in the post-translational control of a yeast transporter.
Volland
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C
Institut J. Monod, Université Paris VII, France.
Garnier
C
C
Haguenauer-Tsapis
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R
D000469:Alkaline Phosphatase / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000302:isolation & purification / Q000378:metabolism*; D033704:Nucleotide Transport Proteins; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins
D000596:Amino Acids; C035528:FUR4 protein, S cerevisiae; D026901:Membrane Transport Proteins; D033704:Nucleotide Transport Proteins; D010750:Phosphoproteins; D029701:Saccharomyces cerevisiae Proteins; D000469:Alkaline Phosphatase