Casein kinase I-dependent phosphorylation within a PEST sequence and ubiquitination at nearby lysines signal endocytosis of yeast uracil permease
Casein kinase I-dependent phosphorylation within a PEST sequence and ubiquitination at nearby lysines signal endocytosis of yeast uracil permease
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DOI：
10.1074/jbc.m001735200
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发表时间：
2000-08-04
影响因子：
4.8
通讯作者：
Urban-Grimal, D
中科院分区：
文献类型：
--
作者：
Marchal, C;Haguenauer-Tsapis, R;Urban-Grimal, D
酿酒酵母对尿嘧啶的摄取由FUR 4编码的尿嘧啶通透酶介导。通过质膜磷酸化修饰尿嘧啶通透酶是调节该蛋白质内吞作用的关键机制。这种修饰反过来又促进了它的泛素化和内化。内吞作用后，通透酶靶向溶酶体/液泡进行蛋白水解。我们以前已经表明，尿嘧啶通透酶是磷酸化的几个丝氨酸残基内的一个很好的特点N-末端PEST序列。在本报告中，我们提供了证据表明，邻近PEST序列的赖氨酸残基38和41是透性酶遍在蛋白化的目标位点。在Lys（38）和Lys（41）处的保守取代产生磷酸化但不能内化的变体渗透酶。PEST序列含有符合酪蛋白激酶1的共有序列的潜在磷酸化位点。酪蛋白激酶1（CK 1）蛋白激酶，由冗余的YCK 1和YCK 2基因编码，位于质膜上。任何一个单独支持生长，但两者的功能丧失都是致命的。在这里，我们表明，在CK 1缺陷的细胞，通透酶磷酸化差，差泛素化。此外，CR 1的过度生产挽救了突变通透酶的内吞缺陷，其中丝氨酸磷酸化受体被苏氨酸（一种不太有效的磷酸化受体）取代，这表明Yck活性可能在通透酶的磷酸化中发挥直接作用。尽管CK 1缺陷细胞中的蛋白质的泛素化水平较低，但渗透酶内化并没有受到很大影响。这可能是由于CK 1在内吞作用中具有第二种抵消作用，如通过具有不可磷酸化形式的PEST序列的变体渗透酶的更高周转所示。
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, CR1 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.