Negative Regulation of the Yeast ABC Transporter Ycf1p by Phosphorylation within Its N-terminal Extension

Abstract

The yeast vacuolar membrane protein Ycf1p and its mammalian counterpart, MRP1, belong to the ABCC subfamily of ATPbinding cassette (ABC) transporters that rid cells of toxic endogenous and xenobiotic compounds. Like most members of the ABCC subfamily, Ycf1p contains an N-terminal extension in addition to its ABC "core" domain and transports substrates in the form of glutathione conjugates. Ycf1p is subject to complex regulation to ensure its optimal function. Previous studies showed that Ycf1p activity is stimulated by a guanine nucleotide exchange factor, Tus1p, and is positively regulated by phosphorylation in its ABC core domain at residues Ser-908 and Thr-911. Here we provide evidence that phosphorylation of Ser-251 in the Ycf1p N-terminal extension negatively regulates activity. Mutant Ycf1p-S251A exhibits increased resistance to cadmium in vivo and increased Ycf1p-dependent transport of [ 3 H]estradiol-␤-17-glucuronide in vitro as compared with wild-type Ycf1p. Activity is restored to the wild-type level for Ycf1-S251E. To identify kinase(s) that negatively regulate Ycf1p function, we conducted an integrated membrane yeast two-hybrid (iMYTH) screen and identified two kinase genes, CKA1 and HAL5, deletion of which increases Ycf1p function. Genetic evidence suggests that Cka1p may regulate Ycf1p function through phosphorylation of Ser-251 either directly or indirectly. Overall, this study provides compelling evidence that negative, as well as positive, regulation of Ycf1p is mediated by phosphorylation.

ABC Transporters in Saccharomyces cerevisiae and Their Interactors: New Technology Advances the Biology of the ABCC (MRP) Subfamily

“…One additional phosphorylated residue, S251, lies within the L0 domain of Ycf1p. Recent studies from our group suggest that phosphorylation at S251 may negatively regulate Ycf1p function, since an S251A mutant exhibits increased resistance to cadmium in vivo and increased Ycf1p transport activity in vitro compared to WT YCF1 (118). Both activities are restored to the WT level in an S251E mutant.…”

“…Intriguingly, deletion mutants of Tus1p, Num1p, and Fab1p displayed reduced resistance to cadmium and arsenite, providing strong evidence that they work in concert with, or positively regulate, Ycf1p function (119). Additional experiments with Tus1p also showed that it appeared to be involved in the formation of the red color in ade2⌬ cells, a phenotype known to be associated with the Ycf1p-mediated uptake of an adenine biosynthetic intermediate into the vacuole (18,118,119,145), as discussed above. Vacuolar uptake experiments using a radiolabeled substrate also revealed that the addition of cytosolic extracts from cells expressing Tus1p stimulated Ycf1p-mediated uptake in a GTP-dependent manner.…”

“…The studies discussed above suggest an important mechanistic role of phosphorylation in both positively and negatively regulating Ycf1p transporter function. Interestingly, many putative Ycf1p phosphorylated residues are highly conserved in human MRPs (118). Thus, findings for Ycf1p phosphorylation at particular sites could pave the way for examining whether mutations that ablate or mimic phosphorylation have a predictable pattern of positive or negative impact on function in mammalian ABCC transporters.…”

“…Notably, through interactor studies (discussed below), a kinase gene, CKA1, that encodes a subunit of the protein kinase, CKII, was identified. Interestingly, a cka1⌬ mutant is cadmium sensitive, and additional genetic evidence points to the possibility that Cka1p may act by phosphorylating S251 of Ycf1p (118). In general, the role of phosphorylation in regulating ABC protein function remains unclear.…”

“…Ycf1p appears to be strongly positively regulated by phosphorylation. Two residues (S908 and T911) within the linker of the core domain of Ycf1p are important for its positive regulation by phosphorylation (36,118). The alteration of either residue to alanine (S908A and T911A) lowers cadmium resistance in vivo and LTC 4 transport in vitro.…”