Casein kinase I-dependent phosphorylation within a PEST sequence and ubiquitination at nearby lysines signal endocytosis of yeast uracil permease

Uracil uptake by Saccharomyces cerevisiae is mediated by the FUR4-encoded u racil permease. The modification of uracil permease by phosphorylation at t he plasma membrane is a key mechanism for regulating endocytosis of this pr otein. This modification in turn facilitates its ubiquitination and interna lization. Following endocytosis, the permease is targeted to the lysosome/v acuole for proteolysis. We have previously shown that uracil permease is ph osphorylated at several serine residues within a well characterized N-termi nal PEST sequence. In this report, we provide evidence that lysine residues 38 and 41, adjacent to the PEST sequence, are the target sites for ubiquit ination 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 p lasma membrane. Either alone supports growth, but loss of function of both is lethal. Here, we show that in CK1-deficient cells, the permease is poorl y phosphorylated and poorly ubiquitinated. Moreover, CR1 overproduction res cued the defective endocytosis of a mutant permease in which the serine pho sphoacceptors were replaced by threonine (a less effective phosphoacceptor) , which suggests that Yck activity may play a direct role in phosphorylatin g the permease. Permease internalization was not greatly affected in CK1-de ficient cells, despite the low level of ubiquitination of the protein. This may be due to CK1 having a second counteracting role in endocytosis as sho wn by the higher turnover of variant permeases with unphosphorylatable vers ions of the PEST sequence.