Abstract
The yeast vacuolar membrane protein Ycf1p and its mammalian counterpart, MRP1, belong to the ABCC subfamily of ATP-binding 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-beta-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.
Highlights
Members of the ABCC subfamily have two distinguishing features in addition to their sequence homology
Whereas all ATPbinding cassette (ABC) transporters contain an ABC “core” domain (two membrane-spanning domains (MSDs) and two nucleotide-binding domains (NBDs) connected by a cytosolic domain; see Fig. 1A), a hallmark of the ABCC subfamily is that most of its members contain a significant N-terminal extension (NTE) [4, 6]
The substrate specificity, biochemical requirements for transporter function, and factors involved in transcriptional regulation of ABCC transporters have been relatively well studied [4, 6, 16, 17]
Summary
Standard dropout media (SC) were prepared as described previously [41]. The double knock-out strains SM5515 (⌬ycf1::URA3 ⌬hal5::KanMX) and SM5516 (⌬ycf1::URA3 ⌬cka1::KanMX) were created by standard homologous recombination as follows. A PCR product containing the URA3 coding sequence flanked by 60 bp of homology to the 5Ј upstream and 3Ј downstream coding sequence of YCF1 was gel-purified according to the manufacturer’s directions (Qiagen), transformed into SM5509 (⌬hal5::KanMX) and SM5510 (⌬cka1::KanMX), and double disruptants were selected on SC-URA G418-containing plates, yielding strains SM5515 and SM5516, respectively. Phosphorylation site mutants of Ycf1p were created in the starting plasmid pSM1753 (2 YCF1-GFP URA3) using standard PCR-mediated mutagenesis (Stratagene QuikChange II-XL). Primers harboring the mutation of interest were designed using the QuikChange Web site primer design
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