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: phosphor ylation 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. I nternalization of the transporter was reduced when some of the serines within the region were converted to alanines and severely impaired wh en all five serines within the region were mutated or when this region was absent. The phosphorylation and degree of ubiquitination of varia nt permeases were inversely correlated with the number of serines repl aced by alanines. A serine-free version of this sequence was very poor ly phosphorylated, and elimination of this sequence prevented ubiquiti nation. Thus, it appears that the serine residues in the PEST-like seq uence are required for phosphorylation and ubiquitination of uracil pe rmease. A PEST-like sequence in which the serines were replaced by glu tamic acids allowed efficient permease turnover, suggesting that the P EST serines are phosphoacceptors.