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.

Uracil uptake by Saccharomyces cerevisiae is mediated by the FUR4-encoded uracil permease. The modification of uracil permease by phosphorylation at the plasma membrane is a key mechanism for regulating endocytosis of this protein. This modification in turn facilitates its ubiquitination and internalization. Following endocytosis, the permease is targeted to the lysosome/vacuole for proteolysis. We have previously shown that uracil permease is phosphorylated at several serine residues within a well characterized N-terminal PEST sequence. In this report, we provide evidence that lysine residues 38 and 41, adjacent to the PEST sequence, are the target sites for ubiquitination of the permease. Conservative substitutions at both Lys 38 and Lys 41 give variant permeases that are phosphorylated but fail to internalize. The PEST sequence contains potential phosphorylation sites conforming to the consensus sequences for casein kinase 1. Casein kinase 1 (CK1) protein kinases, encoded by the redundant YCKI and YCK2 genes, are located at the plasma membrane. Either alone supports growth, but loss of function of both is lethal. Here, we show that in CK1-deficient cells, the permease is poorly phosphorylated and poorly ubiquitinated. Moreover, CK1 overproduction rescued the defective endocytosis of a mutant permease in which the serine phosphoacceptors were replaced by threonine (a less effective phosphoacceptor), which suggests that Yck activity may play a direct role in phosphorylating the permease. Permease internalization was not greatly affected in CK1-deficient cells, despite the low level of ubiquitination of the protein. This may be due to CK1 having a second counteracting role in endocytosis as shown by the higher turnover of variant permeases with unphosphorylatable versions of the PEST sequence.