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 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.
Marchal
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C
Institut Jacques Monod, CNRS-UMRC9922, Université Paris 6 and Paris 7-Denis Diderot, 2 Place Jussieu, 75251 Paris cedex 05, France.
Haguenauer-Tsapis
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R
Urban-Grimal
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D
D000595:Amino Acid Sequence; D001692:Biological Transport; D047389:Casein Kinase I; D047388:Casein Kinases; D004705:Endocytosis; D005800:Genes, Fungal; D014644:Genetic Variation; D006207:Half-Life; D008239:Lysine / Q000235:genetics; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D033704:Nucleotide Transport Proteins; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins / Q000378:metabolism*
C035528:FUR4 protein, S cerevisiae; D026901:Membrane Transport Proteins; D033704:Nucleotide Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; D011494:Protein Kinases; D047389:Casein Kinase I; D047388:Casein Kinases; C479666:YCK2 protein, S cerevisiae; D008239:Lysine