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

EXPERIMENTAL PROCEDURES

Yeast Strains, Media, and Growth Conditions—Yeast strains used in this study are listed in Table 1. Standard dropout media (SC) were prepared as described previously (41). The plates used for cadmium spot tests were prepared by adding cadmium (CdCl₂) to the indicated concentrations in SC medium immediately before pouring plates as described (9, 23). Cultures were grown at 30 °C. The yeast medium used in iMYTH screening is described in Iyer et al. (36).

The double knock-out strains SM5515 (Δycf1::URA3 Δhal5::KanMX) and SM5516 (Δycf1::URA3 Δcka1::KanMX) were created by standard homologous recombination as follows. The single deletions Δhal5::KanMX and Δcka1::KanMX in the BY4741 background were obtained from the MATa yeast genome deletion collection (Open Biosystems). We confirmed the replacement of each gene with the KanMX cassette by standard PCR using a forward primer within the KanMX cassette and a reverse primer within the 3′ region of each gene. Each confirmed deletion was given an SM strain designation (SM5509 and SM5510) to signify that the appropriate genotype was confirmed. A PCR product containing the URA3 coding sequence flanked by 60 bp of homology to the 5′ upstream and 3′ downstream coding sequence of YCF1 was gel-purified according to the manufacturer's directions (Qiagen), transformed into SM5509 (Δhal5::KanMX) and SM5510 (Δcka1::KanMX), and double disruptants were selected on SC-URA G418-containing plates, yielding strains SM5515 and SM5516, respectively.

Plasmids—Plasmids used in this study are listed in Table 2. Phosphorylation site mutants of Ycf1p were created in the starting plasmid pSM1753 (2μ YCF1-GFP URA3) using standard PCR-mediated mutagenesis (Stratagene QuikChange II-XL). In brief, primers harboring the mutation of interest were designed using the QuikChange Web site primer design program (Stratagene), and PCR-mediated mutagenesis was carried out. The product of the PCR reaction was digested for 1–3 h with DpnI. A 10-μl aliquot of the DpnI digest was ligated (Takara Co.) and transformed into the DH5α bacterial cloning strain. DH5α clones containing mutant plasmid constructs were selected on LB agar plates containing carbenicillin, purified, and sequenced for verification, which resulted in the creation of pSM2243, pSM2244, and pSM2245, containing S251A, S251E, and S908A,T911A, respectively. To construct the YCF1 triple mutant, S251A,S908A,T911A, a restriction fragment containing the S251A mutation from pSM2243 was co-transformed with linearized pSM2245 into yeast, and homologous recombination generated the recombinant plasmid pSM2246.

Construction of a NubG-X Yeast cDNA Library and iMYTH Screen with YCF1-CT as Bait—An oligo(dT)-primed, size-selected (0.8–5 kb; average insert size, 1.6 kb) yeast cDNA library (S. cerevisiae, strain Jel1) with 2 × 10⁶ independent clones was custom constructed in the prey vector, pDL2Nx, by Dualsystems Biotech Inc. This cDNA library was transformed into the yeast reporter strain THY AP4 expressing the chromosomally tagged YCF1-CT bait (34). Approximately 8 × 10⁶ transformants were screened for colonies that would grow on media lacking tryptophan (W), adenine (A), and histidine (H) supplemented with 25 mm 3-aminotriazole. Library plasmids were isolated from 102 positive clones, amplified in Escherichia coli, and analyzed by restriction analysis for insert sizes. The plasmids that contained an insert were further processed by a bait dependence test. For this purpose, the individual prey plasmids were retransformed into THY AP4 yeast expressing YCF1-CT and THY AP4 expressing a yeast plasma membrane protein, SHO1-CT, after which 77 YCF1-CT-dependent clones were identified, four of which encoded Cka1p and five of which encoded Hal5p, respectively. The nature of the remaining 68 YCF1-CT-dependent clones will be described elsewhere. Lastly, the remaining 25 clones interacted with both YCF1-CT and SHO1-CT and were therefore considered false positives.

Growth Inhibition by Cadmium—Growth inhibition by CdCl₂ was monitored by spot tests on plates essentially as described previously (9, 23). Briefly, cells were grown overnight to saturation in SC-dropout medium, subcultured at a 1:5000 dilution in the same medium, and grown overnight to an A₆₀₀ of ∼1.0. The overnight culture was diluted to an A₆₀₀ of 0.1, which in turn was diluted in 10-fold increments. Aliquots (4 μl) of each 10-fold dilution were spotted onto SC-dropout plates containing no drug (vehicle alone), 50 μm, or 200 μm CdSO₄ and incubated for 2 days (no drug) or 4 days (50 and 200 μm drug), respectively.

To examine growth inhibition by CdCl₂ in liquid media, cells were grown overnight to saturation in SC medium, subcultured at a 1:5000 dilution in SC medium, and grown overnight to an A₆₀₀ of ∼1.0. The overnight culture was diluted to an A₆₀₀ of 0.1 in YPD medium (yeast/peptone/dextrose) containing 400 μm CdCl₂, or to an A₆₀₀ of 0.02 in YPD alone, and incubated at 25 °C for 20 h. A₆₀₀ was measured and plotted in a bar graph as percent of control ((A₆₀₀ of deletion strain/A₆₀₀ of WT) × 100%).

Immunoblotting Analysis of GFP-tagged Ycf1p—Cell extracts and immunoblots to examine Western blots of WT and mutant Ycf1p were prepared essentially as described previously (22), with modifications as follows. Fifty ml of cells grown in log phase to an A₆₀₀ of 0.8–1.0 units were harvested, resuspended in 100 μl of radioimmune precipitation assay buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1% Nonidet P-40, and 0.5% deoxycholate) containing 1% SDS, fresh protease inhibitors (Roche Complete protease inhibitor mixture), phenylmethylsulfonyl fluoride (1 μm), and pepstatin A (5 μg/ml)), and broken with glass beads (1 min of vortexing and 1 min on ice for 10 times). Following bead beating, 900 μl of radioimmune precipitation assay buffer plus protease inhibitors without SDS was added, and the broken cells were microfuged at 2000 rpm for 5 min at 4 °C to remove debris. The protein concentration in the supernatant was measured by the Bradford assay. For standard Western blot analysis, 12.5 μg of total protein was solubilized in an equal volume of 2× SDS-PAGE sample buffer (50 mm Tris-HCl, pH 6.8, 8 m urea, 0.1 mm EDTA, 15% SDS, 0.01% bromphenol blue, and 150 mm dithiothreitol added fresh), frozen overnight, and subsequently incubated at 37 °C for 1 h prior to SDS-PAGE and transfer to nitrocellulose. For Western blot analysis of GFP-tagged Ycf1p from total protein extracts, blots were incubated with the primary monoclonal mouse anti-GFP (1:1000) (Roche Applied Science) or polyclonal rabbit anti-hexokinase (1:200,000) (courtesy of Dr. Rob Jensen) and the respective secondary antibodies, sheep anti-mouse (1:2000) and sheep anti-rabbit (1:5000). Protein was visualized and quantitated using a chemiluminescent reagent (Roche Applied Science) on a Bio-Rad Versadoc imaging system.

In Vitro Transport of Radiolabeled Substrate by Ycf1p into Vesiculated Vacuoles—To assay transport activity of Ycf1p in vitro, vacuoles were isolated and purified essentially as described previously (34, 42). Ycf1p-dependent transport into the purified vacuoles was assayed by measuring the amount of radiolabeled Ycf1p substrate, [³H]estradiol-β-17-glucuronide ([³H]E₂β17G), transported from outside to inside the vesiculated vacuoles. Transport assays were conducted as described previously (34) with minor modification as follows. 100-μl reaction volumes contained NTPs, NaATP, or NaAMP-PCP (at 4 mm); 10 mm MgCl₂;5 μm gramicidin-D; 10 mm creatine phosphate and 16 units/ml creatine kinase; 50 mm KCl; 400 mm sorbitol; 25 mm Tris-Mes (pH 8.0); and a Ycf1p substrate, [³H]E₂β17G (Amersham Biosciences), at concentrations of 50, 100, 200, 400, 800, 1600, and 2400 μm. Reactions were initiated by the addition of 1–2 μg of purified vacuoles and incubated at 25 °C for 5 or 10 min; they were stopped by the addition of 1 ml of ice-cold stop buffer (400 mm sorbitol, and 3 mm Tris/Mes (pH 8.0)). Vacuoles were collected on Millipore 0.22-μm GV filters via vacuum manifold and washed. Transport was measured as total dpm, normalized to Ycf1p protein levels as determined by Western blot (described above), and reported as nmol of substrate ([³H]E₂17G) sequestered within the vacuoles/mg of total protein over 10 min (34).

The efficiency of transport for Ycf1p, Ycf1p-S251A, Ycf1p-S251E, and Ycf1p-S908A,T911A was analyzed by standard Michaelis-Menten kinetics in Kaleidagraph (Synergy Software). V_max and K_mapp values of E₂β17G transport for each protein were extrapolated from the curve. Transport efficiency (see Table 3) is reported as V_max/K_mapp, and relative transport efficiency of mutants is expressed as (V_max/K_mapp of mutant/V_max/K_mapp of WT).

Fluorescence Microscopy—To examine the localization of Ycf1p-GFP fusion proteins, cells were grown overnight to saturation in minimal medium and then subcultured at a 1:1000 dilution in minimal medium and grown overnight in log phase to an A₆₀₀ of ∼0.7. Cells were examined at ×100 magnification on polylysine-coated slides using an Axioskop microscope equipped with fluorescence and Nomarski optics (Carl Zeiss, Thornwood, NY). Images were captured with a Cooke charge-coupled device camera and IP Lab Spectrum Software (Biovision Technologies, Exton, PA) (9, 23, 34).

RESULTS

Phosphorylation of Serine 251 Negatively Regulates Ycf1p Function in Vivo—Ycf1p function is known to be positively regulated by phosphorylation at Ser-908 and Thr-911 within the ABC core (Fig. 1A) (35). Among phosphopeptides reported in recent high throughput yeast screens were two that contained a novel phosphorylation site at Ser-251 within the NTE of Ycf1p (Fig. 1A) (39, 40). To determine if phosphorylation at Ser-251 regulates Ycf1p function, we generated a Ycf1p-S251A mutation to inhibit phosphorylation and a Ycf1p-S251E mutation to mimic phosphorylation.

C-terminally GFP-tagged versions of these mutants were expressed in a Δycf1 strain background along with WT Ycf1p and the previously described phosphorylation mutant Ycf1p-S908A,T911A (35). All of these proteins are expressed roughly the same expression level and correctly localized to the vacuole membrane (Fig. 1, B and C, respectively). The somewhat diminished expression of Ycf1p-S908A,T911A seen here (∼50% of WT; Fig. 1B, compare lanes 2 and 5) can also be inferred from a study published by others (35). A slight mobility shift is also evident for the Ycf1p-S908A,T911A mutant protein, as was observed previously. The mutation of S251A did not result in a detectable SDS-PAGE mobility shift for full-length Ycf1p or the N-terminal fragment4; however, a mobility shift due to phosphorylation is often not detectable (43–45).

We examined the ability of strains expressing GFP-tagged WT and mutant Ycf1p to grown on plates containing cadmium. Surprisingly, the Ycf1p-S251A mutant is more resistant to cadmium than the strain expressing WT Ycf1p (Fig. 2A, compare rows 1 and 3). This result is the opposite of that from the Ycf1p-S908A,T911A mutant (Fig. 2A, compare rows 1 and 5), which is more sensitive to cadmium than the WT but not as sensitive as the Δycf1 strain (row 2). The resistance to cadmium associated with the expression of Ycf1p-S251A is reversed to approximately that of the WT (Ser-251) level in a strain expressing Ycf1p-S251E (Fig. 2A, compare rows 1 and 4). The increased cadmium resistance observed for the Ycf1p-S908A,T911A mutant is not altered by the addition of the S251A mutation (Fig. 2B, compare rows 4 and 5), suggesting that regulation of Ycf1p activity via phosphorylation of Ser-251 is dependent upon the functionality imparted by phosphorylation of Ser-908 and Thr-911.

We also carried out these studies in liquid media in which cadmium was present and observed an analogous relationship in the growth of the single and double mutant strains (Fig. 2C). These results suggest that phosphorylation of Ser-251 negatively regulates Ycf1p function in vivo.

Phosphorylation of Ycf1p at Residue Ser-251 Negatively Regulates Transport Activity in Vitro—To determine whether the increased cadmium resistance of the Ycf1p-S251A mutant is directly due to increased Ycf1p function, we measured Ycf1p transport activity using an in vitro transport assay and a well characterized Ycf1p substrate, E₂β17G (34). Assays were performed with vesiculated vacuoles derived from strains expressing no Ycf1p, WT Ycf1p, and various Ycf1p phosphorylation site mutants. The results shown in Fig. 3 reflect the in vivo Cd²⁺ resistance findings. Vacuoles derived from Ycf1p-S251A strains have greater transport activity than WT, whereas Ycf1p-S251E-derived vacuoles have transport activity similar to that of WT (Fig. 3A). These findings further support the conclusion that phosphorylation at Ser-251 promotes negative regulation of Ycf1p function. In comparison, vacuoles derived from a Ycf1p-S908A,T911A strain have decreased transport activity, in agreement with what has been reported previously (35).

To gain insight into the mechanism by which phosphorylation of Ser-251 regulates Ycf1p transporter activity, we determined the Michaelis-Menten transport kinetics of the WT and mutant Ycf1p proteins (Fig. 3B and Table 3). Ycf1p-S251A is ∼2 fold more efficient in its ability to transport E₂β17G than WT (Table 3, last column), largely because of a change in the K_mapp for substrate binding. This trend is reversed by the Ycf1p-S251E mutation. Ycf1p-S908A,T911A kinetics have not been reported previously. Here we show this mutation is ∼2.5 fold less efficient for transport than that of WT (Fig. 3B and Table 3, last column). The decrease in transport efficiency for Ycf1p-S908A,T911A is the result of both an increase in K_mapp for the substrate (E₂β17G) and a decrease in V_max,app as compared with WT (Fig. 3B and Table 3). Together these experiments suggest that phosphorylation in different regions within Ycf1p has opposing effects: phosphorylation of Ser-251 in the NTE appears to negatively regulate Ycf1p function, whereas phosphorylation of Ser-908 and Thr-911 in the ABC core positively regulates Ycf1p function.

Identification of Hal5p and Cka1p as Ycf1p Protein Interactors by iMYTH—To seek a kinase(s) that could be involved in the negative regulation of Ycf1p, we used a modified yeast two-hybrid “split ubiquitin” screen called the integrated membrane yeast two-hybrid assay. iMYTH is specifically designed for detecting membrane protein interactors and has proven extremely useful in identifying the physiologically relevant Ycf1p interactor, Tus1p, which positively regulates Ycf1p transport activity (34). Complete details of the iMYTH screening procedure are provided under “Experimental Procedures” and elsewhere (34). In the library used here, and in contrast to our recently published work (34), cDNA fragments of ∼0.8 to 5 kb in length were inserted C-terminally to the NubG sequence, generating the library in NubG-X orientation (where X is a cDNA insert). This library was introduced by transformation into the yeast strain THY AP4-YCF1-CT expressing the endogenously tagged YCF1-CT “bait.” From an iMYTH screen of ∼8 × 10⁶ yeast transformants, 18 novel Ycf1p interactors were identified, among which two new kinases were found (Fig. 4A). One of these is Cka1p, a catalytic component of the yeast multisubunit casein kinase II (CKII) (46), and the other is Hal5p, a Ser/Thr kinase associated with yeast halotolerance (Fig. 4A) (47). In this study, we focused our attention on these two new kinases; the analysis of the remaining 16 newly identified Ycf1p interactors will be described elsewhere.

Deletion of Hal5p or Cka1p Results in Increased Cadmium Resistance in Vivo and Increased Transport Activity in Vitro—To determine whether the deletion of the CKA1 and HAL5 genes affects cadmium resistance, we carried out growth tests by spot dilution on yeast agar plates containing 50 μm Cd²⁺ (Fig. 4B). We observed a significant increase in cadmium resistance for both the Δhal5 and the Δcka1 mutants (Fig. 4B, compare rows 3 and 4 with row 1). Increased resistance is dependent upon Ycf1p, as it does not occur in the Δycf1 Δhal5 and Δycf1 Δcka1double mutant strains (Fig. 4B, compare rows 5 and 6 with rows 3 and 4). These experiments indicate that Hal5p and Cka1p either directly or indirectly negatively regulate Ycf1p function.

To test whether the increased cadmium resistance observed in the Δhal5 and Δcka1 mutants in vivo is due to increased Ycf1p function, we conducted in vitro transport assays with the Ycf1p substrate [³H]E₂β17G and vesiculated vacuoles derived from the Δhal5 and Δcka1 strains (Fig. 4C). If Hal5p and Cka1p kinases are negative regulators of Ycf1p function, then vacuoles derived from Δhal5 and Δcka1 deletion strains should exhibit increased Ycf1p-dependent transport. This is indeed the case, as shown in Fig. 4C. Notably, the increased activity observed for the kinase deletions is similar to the increased activity observed for strains expressing the Ycf1p-S251A mutant. Importantly when Ycf1p is absent in the Δhal5 and Δcka1 strains (Fig. 4C, ycf1 hal5 and ycf1 cka1), the kinase deletions show almost no transport activity, indicating that the increased activity observed for Δhal5 and Δcka1 is Ycf1p-dependent and not due to activation of another transporter. These experiments indicate that the Hal5p and Cka1p kinases negatively regulate Ycf1p activity.

To examine whether Hal5p and/or Cka1p regulate Ycf1p through phosphorylation of Ser-251, the phenotypes of the hal5Δ and cka1Δ strains expressing WT and mutant forms of Ycf1p were examined on cadmium-containing plates (Fig. 4D). Interestingly, in the cka1Δ strain, the cadmium resistance of WT Ycf1p is indistinguishable from that of Ycf1p-S251A (Fig. 4D, rows 1 and 2), possibly because WT Ycf1p cannot be phosphorylated at Ser-251 in the cka1Δ mutant, and thus negative regulation is lifted. In contrast, the kinase-independent mutant Ycf1p-S251E shows reduced cadmium resistance (Fig. 4D, row 3). These results are consistent with the possibility that Cka1p could negatively regulate Ycf1p function through phosphorylation of Ser-251, either directly or indirectly. In contrast, growth of the hal5Δ strains expressing either WT or mutant Ycf1p constructs grew similarly on cadmium, suggesting that Hal5p does not act through Ser-251 (Fig. 4D, lower panel, rows 4–6).

DISCUSSION

The ABCC transporter Ycf1p is critical for the detoxification of heavy metals in yeast. One of our long-term goals is to understand how Ycf1p activity is regulated. Like other members of the ABCC subfamily of transporters, including MRP1 and MRP2 in mammalian cells, Ycf1p contains an NTE with five membrane spans (MSD0) and a cytosolic loop (L0) in addition to its ABC core transporter domain (48, 49). Our studies and those of others have shown that L0 plays a key role in activity, because when L0 is deleted, Ycf1p, ABCC1 (MRP1), and ABCC2 (MRP2) are properly localized but exhibit no transport activity (8–10). This finding is somewhat surprising, as the ABC core suffices for the activity of most other ABC transporters, and it suggests a regulatory role for the L0 domain in the ABCC subfamily (4, 50). Two recent high throughput study identified residue Ser-251 in L0 of Ycf1p as a site of phosphorylation (39, 40). In the present study we have provided evidence that phosphorylation of Ser-251 negatively regulates Ycf1p activity. We show that cells expressing the mutant protein Ycf1p-S251A, in which phosphorylation should be abrogated, exhibited higher Ycf1p activity than cells expressing WT Ycf1p, both in vivo (cadmium resistance) and in vitro (E₂β17G transport). On the other hand, cells expressing Ycf1-S251E, expected to mimic the phosphorylated form, exhibited wild-type activity. Thus, phosphorylation of L0 within the NTE appears to negatively regulate Ycf1p, as indicated in the model shown in Fig. 5A, and when phosphorylation is absent, the activity of Ycf1p increases (Fig. 5B).

Previous studies have shown that phosphorylation within the ABC core of Ycf1p at residues Ser-908 and Thr-911 positively regulates Ycf1 function (26, 35). Mutation of both sites to alanine decreases cadmium resistance and Ycf1p-dependent transport activity, a result recapitulated in this study. Thus, Ycf1p appears to be highly regulated by phosphorylation, both negatively, in the NTE, and positively, in the ABC core domain.

The L0 domain of the NTE is critical for ABCC function (7, 11, 51). MRP1 cross-linking studies suggest that L0 contributes to substrate binding, either directly or indirectly, by facilitating an interaction of substrate and the ABC core (10, 14). Phosphorylation of L0 at Ser-251 could inhibit substrate interaction with the Ycf1p core, accounting for the decrease in K_m that we observed here for the S251A mutant. The ability of the L0 to interact with the membrane via two predicted amphipathic helices is required for MRP1 function. These amphipathic helices are conserved throughout the ABCC subfamily, including Ycf1p. Ser-251 of Ycf1p lies at the N-terminal proximal end of amphipathic helix 2 (11). Further, this region of L0 is required for homodimerization of MRP1. The inability of MRP1 to form a dimer appears to be required for function (15). Therefore, it is tempting to speculate that phosphorylation of Ser-251 could inhibit the interaction of amphipathic helix 2 with the membrane, thereby decreasing protein function. Alternatively, phosphorylation of Ser-251 may inhibit homodimerization, resulting in decreased Ycf1p function. In contrast, we observed an increased K_m and decreased V_max of transport for the S908A,T911A double mutant, suggesting that phosphorylation within the ABC core could negatively affect both substrate binding and transport.

We were interested in identifying the kinases that regulate Ycf1p activity. To do so, we used iMYTH technology, which we have previously shown to be an extremely effective method of identifying interactors (i.e. Tus1p) of membrane proteins such as Ycf1p (34). Here, we screened among kinase interactors for those that negatively regulate transporter function in a manner similar to phosphorylation of Ser-251. In this way, we identified genes encoding the kinase Hal5p and the catalytic subunit Cka1p of the serine/threonine kinase CKII (46). Hal5p plays a role in salt and pH tolerance in yeast through regulating the trafficking of the potassium transporters Trk1p and Trk2p (47, 52). CKII is involved in multiple processes, including the cell cycle, cell polarization, and transcription regulation (53). The residues flanking Ser-251 in Ycf1p conform to the CKII consensus sequence, which is conserved from yeast to humans (Fig. 6) Furthermore, the L0 region of the human ABCC6 (MRP6) shares strong homology with that of Ycf1p (11), including Ser-244 in ABCC6, which is positionally conserved with Ser-251 in Ycf1p. Both lie within a CKII consensus sequence, and Ser-251 of Ycf1p and Ser-244 in ABCC6 were predicted to be sites of CKII phosphorylation by the programs Scansite and Group-based Phosphorylation Scoring (GPS) (Fig. 6) (54–57). It will therefore be interesting to determine whether mutation of Ser-244 impacts ABCC6 function. Mutations in Ser-244 have not been associated with pseudoxanthoma elasticum (58); however, an interesting possibility is that mutations at this site could potentially modify the severity of pseudoxanthoma elasticum phenotypes.

The single deletion mutants Δhal5 and Δcka1 exhibited increased resistance to cadmium in vivo as compared with WT cells; vacuoles derived from these mutants have increased Ycf1p-dependent transport activity in vitro, a pattern similar to a strain expressing Ycf1p-S251A. These results suggest that the Hal5p and CKII kinases negatively regulate Ycf1p function, but they could do so either by similar or distinct mechanisms. Further genetic evidence (Fig. 4) provides support for the possibility that Cka1p may directly or indirectly phosphorylate Ser-251, as WT Ycf1p function is indistinguishable from Ycf1p-S251A in a cka1Δ strain as measured by cadmium resistance. However, because we currently cannot directly measure phosphorylation of Ser-251, it would be premature to draw the conclusion that Cka1p phosphorylates this residue. It is certainly possible that Cka1p does not phosphorylate Ycf1p directly but instead influences an as yet unknown factor that is the direct regulator of Ycf1p phosphorylation or in some other way negatively impacts Ycf1p function (but does not affect the expression level or vacuolar localization of Ycf1p). Taken together, the results presented here and elsewhere suggest an important mechanistic role of phosphorylation in negatively and positively regulating Ycf1p transporter function. Future studies will focus on directly establishing which kinases mediate phosphorylation at specific sites in Ycf1p and developing a complete phosphoprotein map of Ycf1p.