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 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.

FIGURE 1.

Ycf1p is phosphorylated at Ser-251. A, the structure of Ycf1p consists of an NTE containing membrane-spanning domain MSD0 and a cytosolic linker (L0) and an ABC core region containing two membrane-spanning domains (MSD1 and MSD2) and two nucleotide-binding domains (NBD1 and NBD2). Ycf1p undergoes processing in vivo at the site indicated by an asterisk in MSD1, giving rise to N- and C-terminal fragments that together are required for Ycf1p activity (9). Ycf1p phosphorylation at Ser-908 and Thr-911 is required for transport activity (35), and phosphorylation within the NTE at Ser-251 negatively regulates transport activity, as shown in this study. B, WT and mutant forms of GFP-tagged Ycf1p were examined by Western blot analysis using anti-GFP antibody, and hexokinase was probed with an anti-hexokinase antibody as the loading control. The C-terminal portion of Ycf1p (labeled Ycf1p) contains the GFP tag, as shown in A, and is detected in this analysis. Ycf1p expression was quantitated and normalized to hexokinase expression using ImageQuant software and the Bio-Rad Versidoc system. The amount of Ycf1p expressed relative to WT for S251A, S251E, and S908A,T911A is 1, 1.1, and 0.47, respectively C, vacuole localization of GFP-tagged versions of WT and mutant Ycf1p was examined by fluorescence microscopy. Yeast strains used in B and C are SM5270, SM5280, SM5506, SM5507, and SM5508.

FIGURE 2.

Ycf1p-S251A exhibits increased resistance to cadmium as compared with WT Ycf1p. The ability of strains expressing WT and mutant forms of Ycf1p to grow on CdCl₂-containing SC plates was examined by spot dilution (A and B) or by growth in liquid media containing CdCl₂ (C), as described under “Experimental Procedures.” Tests were performed in triplicate, and error bars are indicated. Yeast strains used were SM4460, SM5270, SM5280, SM5506, SM5507, SM5508, and SM5546.

FIGURE 3.

Ycf1p-S251A exhibits enhanced transport activity in vitro as compared with WT Ycf1p. A, Ycf1p-dependent transport of [³H]E₂β17G into vacuoles derived from strains expressing WT and mutant Ycf1p was measured as described under “Experimental Procedures.” Experiments were conducted in triplicate or quintuplet, and error bars represent mean ± S.D. B, Michaelis-Menten kinetics of Ycf1p-dependent transport of [³H]E₂β17G by vacuoles derived from strains expressing WT Ycf1p (open circle), Ycf1p-S251A (open square), Ycf1p-S251E (closed circle), and Ycf1p-S908A,T911A (closed square) were measured as described under “Experimental Procedures.” Values for K_mapp, V_max,_app, and relative transport efficiency (V_max,app/K_mapp) were extrapolated from plots of transport assays and are shown in Table 3. All data points were calculated from an n = 3 or 4, and error bars represent mean ± S.D. Yeast strains used were SM5270, SM5280, SM5506, SM5507, and SM5508.

FIGURE 4.

Identification of Ycf1p-protein interactors, Hal5p and Cka1p, by iMYTH. Deletion of Hal5p and Cka1p resulted in increases transport by Ycf1p, similar to Ycf1p-S251A. A, iMYTH identifies Hal5p and Cka1p kinases as novel Ycf1p-specific interactors. The yeast reporter strain THY AP4, expressing either YCF1-CT (left) or SHO1-CT (right) bait constructs from the chromosome, was transformed with the indicated (TRP1 marked) NubG prey plasmids. The growth of yeast cells expressing the YCF1-CT and SHO1-CT chimeras with the indicated NubG fusions was monitored on agar plates lacking tryptophan (SD-W, left) and tryptophan, adenine, and histidine (SD-WAH, middle). Three independent colonies were pinned onto SD-W- and SD-WAH-selective plates prior to assessment of β-galactosidase activity using an 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) plate test (right). Both Cka1p and Hal5p kinases show specific activation of the reporter gene system only in the presence of YCF1-CT bait but not in the presence of an unrelated yeast plasma membrane protein, Sho1p, fused to the C terminus of ubiquitin followed by an artificial transcription factor, LexA-VP16, collectively called SHO1-CT. Please note that the control construct (Ost1-NubI) is designed to activate the yeast reporter system independently of any protein-protein interaction because of the high affinity of NubI to associate with the C terminus of ubiquitin and form active ubiquitin irrespective of a protein-protein interaction. The OST1-Nub control indicates that the YCF1-CT and SHO1-CT chimeric bait proteins are expressed and the proteins are properly inserted into the membrane. B, growth of the indicated strains on plates containing CdCl₂ was examined by spot dilution assay. Yeast strains used are SM5270, SM5509, SM5510, SM5515, and SM5516. C, in vitro transport activity of vacuoles derived from the indicated strains was measured as described for Fig. 3A and under “Experimental Procedures.” Experiments were conducted in triplicate or quintuplet, and error bars represent mean ± S.D. Yeast strains used in this experiment are SM5270, SM5511, SM5512, SM5513, and SM5514. D, growth of the indicated strains on plates containing CdCl₂ was examined by spot dilution assay. Yeast strains used are SM5519, SM5520, SM5521, SM5522, SM5523, and SM5524.

FIGURE 5.

Hal5p, Cka1p, and phosphorylation at Ser-251 negatively regulate Ycf1p function. A, in a WT strain, Ycf1p is negatively regulated by phosphorylation at Ser-251 within the L0 domain of the NTE, which diminishes Ycf1p function (thin arrow, weak transport). In addition, Ycf1p interacts with the Cka1p subunit of CKII and the Hal5p kinase, which also negatively regulate Ycf1p function. These kinases could negatively regulate Ycf1p via phosphorylation at Ser-251; genetic evidence suggests that this may be the case for Cka1p (see Fig. 4D). Alternatively, they may act in an as yet undetermined way to decrease Ycf1p function, as discussed in the text. B and C, mutation of Ser-251 to alanine or deletion of the Ycf1p kinase interactor genes HAL5 and CKA1 results in increased Ycf1p function (thick arrow, strong transport). Apparently, in both cases, negative regulation is abolished.

FIGURE 6.

A CKII consensus that contains Ycf1p-Ser-251 is conserved from yeast to humans. The protein sequence of Ycf1p from S. cerevisiae, human ABCC6 (MRP6), and Ycf1p from Saccharomyces bayanus were aligned using ClustalW. Conserved residues have been highlighted using BOXSHADE. The arrow indicates Ser-251 in Ycf1p and the conserved homologous residues in the alignment. The residues surrounding Ser-251 fit a CKII consensus sequence, which is shown below the alignment.