A PEST-like sequence mediates phosphorylation and efficient ubiquitination of yeast uracil permease.
Uptake of uracil by the yeast Saccharomyces cerevisiae is mediated by a specific permease encoded by the FUR4 gene. Uracil permease located at the cell surface is subject to two covalent modifications: phosphorylation and ubiquitination. The ubiquitination step is necessary prior to permease endocytosis and subsequent vacuolar degradation. Here, we demonstrate that a PEST-like sequence located within the cytoplasmic N terminus of the protein is essential for uracil permease turnover. Internalization of the transporter was reduced when some of the serines within the region were converted to alanines and severely impaired when all five serines within the region were mutated or when this region was absent. The phosphorylation and degree of ubiquitination of variant permeases were inversely correlated with the number of serines replaced by alanines. A serine-free version of this sequence was very poorly phosphorylated, and elimination of this sequence prevented ubiquitination. Thus, it appears that the serine residues in the PEST-like sequence are required for phosphorylation and ubiquitination of uracil permease. A PEST-like sequence in which the serines were replaced by glutamic acids allowed efficient permease turnover, suggesting that the PEST serines are phosphoacceptors.
Marchal
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Institut Jacques Monod, CNRS-UMRC9922, Université Paris 7-Denis Diderot, France.
Haguenauer-Tsapis
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Urban-Grimal
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D001665:Binding Sites; D004789:Enzyme Activation / Q000235:genetics; D005800:Genes, Fungal; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D033704:Nucleotide Transport Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017421:Sequence Analysis
C035528:FUR4 protein, S cerevisiae; D026901:Membrane Transport Proteins; D033704:Nucleotide Transport Proteins; D029701:Saccharomyces cerevisiae Proteins