ABCMdb

ABCC2 p.Ser908Ala

Predicted by SNAP2:	A: N (72%), C: N (61%), D: N (66%), E: N (61%), F: N (53%), G: N (66%), H: N (82%), I: N (53%), K: N (72%), L: N (53%), M: N (66%), N: N (78%), P: D (53%), Q: N (78%), R: N (72%), T: N (78%), V: N (57%), W: D (71%), Y: D (66%),
Predicted by PROVEAN:	A: N, C: D, D: N, E: N, F: N, G: N, H: N, I: D, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, T: N, V: N, W: D, Y: N,

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[hide] Suppression of Ycf1p function by Cka1p-dependent p... FEMS Yeast Res. 2010 Nov;10(7):839-57. doi: 10.1111/j.1567-1364.2010.00677.x. Epub 2010 Aug 31. Pickin KA, Ezenwajiaku N, Overcash H, Sethi M, Knecht MR, Paumi CM
Suppression of Ycf1p function by Cka1p-dependent phosphorylation is attenuated in response to salt stress.
FEMS Yeast Res. 2010 Nov;10(7):839-57. doi: 10.1111/j.1567-1364.2010.00677.x. Epub 2010 Aug 31., [PMID:20812950]

Abstract [show]
The yeast vacuolar membrane protein Ycf1p and its mammalian counterpart, MRP1, belong to the ABCC subfamily of ATP-binding cassette transporters. Genetic evidence suggests that the yeast casein kinase 2alpha, Cka1p, negatively regulates Ycf1p function via phosphorylation of Ser251 within the N-terminus. In this study, we provide strong evidence that Cka1p regulates Ycf1p function via phosphorylation of Ser251. We show that the CK2 holoenzyme interacts with Ycf1p. However, genetic analysis suggests that only Cka1p is required for Ser251 phosphorylation, as the deletion of CKA1 significantly reduces Ser251 phosphorylation in vivo. Furthermore, purified recombinant Cka1p phosphorylates a Ycf1p-derived peptide containing Ser251. We also demonstrate that Ycf1p function is induced in response to high salt stress. Induction of the Ycf1p function strongly correlates with reduced phosphorylation of Ser251. Importantly, Cka1p activity in vivo is similarly reduced in response to salt stress, consistent with our finding that Cka1p directly phosphorylates Ser251 of Ycf1p. We provide genetic and biochemical evidence that strongly suggests that the induction of Ycf1p function is the result of decreased phosphorylation of Ser251. In conclusion, our work demonstrates a novel biochemical role for Cka1p regulation of Ycf1p function in the cellular response of yeast to salt stress.

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84 Yeast strains used in this study StrainÃ Relevant genotype Reference CP59w met3Dleu2Dura3Dhis3D Open biosystems CP60 met3Dleu2Dura3Dhis3Dycf1<KanMX Open biosystems CP105 ura3Dade2DLEU2 TRP1 YCF1 This study CP106 ura3DADE2 LEU2 TRP1 YCF1 This study CP135 met15Dleu2Dura3Dhis3Dycf1D<KanMx [2m YCF1-GFP URA3] This study CP136 met15Dleu2Dura3Dhis3Dycf1D<KanMx [2m YCF1-Ser251Ala -GFP URA3] This study CP147 met15Dleu2Dura3Dhis3Dcka1D<KanMx Open biosystems CP151 met15Dleu2Dura3Dhis3Dcka1D<KanMX [2m YCF1-GFP URA3] This study CP156 met15Dleu2Dura3Dhis3DYCF1:TAP-HIS3 Open biosystems CP157 met15Dleu2Dura3Dhis3Dycf11D<KanMX cka1D<URA3 Paumi et al. (2009) CP159 met15Dleu2Dura3Dhis3Dcka2D<KanMX Open biosystems CP164 met15Dleu2Dura3Dhis3DYCF1:TAP-HIS3 KanMx-PGAL-GST-CKA1 This study CP165 met15Dleu2Dura3Dhis3Dycf1D<KanMX [2m YCF1-S869A-GFP URA3] This study CP166 met15Dleu2Dura3Dhis3Dycf1D<KanMX [2m YCF1-S870A -GFP URA3] This study CP167 met15Dleu2Dura3Dhis3Dycf1D<KanMX [2m YCF1-S872A -GFP URA3] This study CP168 met15Dleu2Dura3Dhis3Dycf1D<KanMX [2m YCF1-S873A-GFP URA3] This study CP169 met15Dleu2Dura3Dhis3Dycf1D<KanMX [2m YCF1-S903A-GFP URA3] This study CP170 met15Dleu2Dura3Dhis3Dycf1D<KanMX [2m YCF1-S914A-GFP URA3] This study CP174 PYES2/CT<cka1<v5<6his Kubinski et al. (2007) CP176 met15Dleu2Dura3Dhis3Dcka2D<KanMX [2m CKA2:MYC:6xHis LEU2] This study CP177 met15Dleu2Dura3Dhis3DpepD<KanMX [2m YCF1-GFP URA3] This study CP178 met15Dleu2Dura3Dhis3Dcka2D<KanMX [2m YCF1-GFP URA3] This study CP179 met15Dleu2Dura3Dhis3DKanMx-PGAL-GST-CKA1 This study CP180 met15Dleu2Dura3Dhis3DpepD<KanMX [2m YCF1-Ser251Ala-GFP URA3] This study CP187 met15Dleu2Dura3Dhis3Dcka1D<LEU2 pep4D<KanMX [2m YCF1-TAP URA3] This study CP188 met15Dleu2Dura3Dhis3Dpep4D<KanMX [2m YCF1-TAP URA3] This study CP189 met15Dleu2Dura3Dhis3Dpep4D<KanMX [2m YCF1-Ser869Ala-GFP URA3] This study CP190 met15Dleu2Dura3Dhis3Dpep4D<KanMX [2m YCF1-Ser870Ala-GFP URA3] This study CP191 met15Dleu2Dura3Dhis3Dpep4D<KanMX [2m YCF1-Ser872Ala-GFP URA3] This study CP192 met15Dleu2Dura3Dhis3Dpep4D<KanMX [2m YCF1-Ser873Ala-GFP URA3] This study CP193 met15Dleu2Dura3Dhis3Dpep4D<KanMX [2m YCF1-Ser903Ala-GFP URA3] This study CP194 met15Dleu2Dura3Dhis3Dpep4D<KanMX [2m YCF1-Ser914Ala-GFP URA3] This study CP195 met15Dleu2Dura3Dhis3DCKA1-V5-His<KanMx Open biosystems CP200 met15Dleu2Dura3Dhis3Dpep4D<KanMX [2m YCF1-Ser908Ala,Thr911Ala-GFP URA3] This study CP224 met15Dleu2Dura3Dhis3Dckb1D<KanMX Open biosystems CP225 met15Dleu2Dura3Dhis3Dckb2D<KanMX Open biosystems CP226 met15Dleu2Dura3Dhis3DYCF1:TAP-HIS3 [2m PGAL-GST-CKA2 URA3] This Study CP227 met15Dleu2Dura3Dhis3DYCF1:TAP-HIS3 [2m PGAL-GST-CKB1 URA3] This study CP228 met15Dleu2Dura3Dhis3DYCF1:TAP-HIS3 [2m PGAL-GST-CKB2 URA3] This study CP229 met15Dleu2Dura3Dhis3Dckb1D<KanMX [2m YCF1-GFP URA3] This study CP230 met15Dleu2Dura3Dhis3Dckb2D<KanMX [2m YCF1-GFP URA3] This study CP239 met3Dleu2Dura3Dhis3Dycf1<KanMX [2m YCF1 -GFP URA3] This study CP240 met3Dleu2Dura3Dhis3Dycf1<KanMX [2m YCF1 -Ser251Ala-GFP URA3] This study CP241 met3Dleu2Dura3Dhis3Dycf1<KanMX [2m YCF1 -Ser251Glu-GFP URA3] This study ÃAll CP strains listed here are isogenic to CP59. Login to comment
156 Previously, Eraso et al. (2004) have shown that mutation of Ser908 and Thr911 to alanine resulted in decreased growth on cadmium and decreased Ycf1p-dependent transport activity. Login to comment
161 Additionally, we found that the strain expressing Ycf1p-Ser903Ala has a decreased ability to grow on cadmium (Fig. 2a, compare rows 1 and 8), a phenotype similar to that of the Ycf1p-Ser908Ala, Ycf1p-Thr911Ala, and the Ycf1p-Ser908Ala, Thr911Ala double mutant (Eraso et al., 2004). Login to comment
172 The phenotype of Ycf1p-Ser872Ala and Ycf1p-Ser903Ala is similar to the phenotype shown here for the double mutant Ycf1p-Ser908Ala,Thr911Ala and the phenotype reported previously for the single mutants Ycf1p-Ser908Ala and Ycf1p-Thr911Ala, and the double mutant Ycf1p-Ser908Ala,Thr911Ala (Eraso et al., 2004). Login to comment
192 However, we found one exception, the double mutant Ser908Ala,Thr911Ala. Login to comment
193 It has been shown previously that phosphorylation of Ycf1p-Ser908,Thr911 is required for Ycf1p transport activity and a Ycf1p-Ser908Ala,Thr911Ala mutant is a dominant mutation over the Ser251Ala mutation (Eraso et al., 2004; Paumi et al., 2008). Login to comment
200 We used this strain to generate a YCF1-TAP, GST-CKA1 double-tag strain for subsequent coimmunoprecipitation analysis (described (b) (a) IP: α-GFP WB: α-Ycf1p-S251-P IP: α-GFP WB: α-GFP IP: α-GFP WB: α-Ycf1p-S251-P IP: α-GFP WB: α-GFP IP: α-GFP WB: α-GFP IP: α-GFP WB: α-Ycf1p-S251-P IP: α-GFP WB: α-GFP IP: α-GFP WB: α-Ycf1p-S251-P (c) S908A,T911A Fig. 3. Login to comment
342 To this end, we synthesized a phosphospecific antibody for Ycf1p-Ser251 and showed that linker domain phosphorylation sites do not alter Ycf1p-Ser251 phosphorylation status, with the exception of the Ycf1p-Ser908Ala, Thr911Ala double mutant. Login to comment
347 Interestingly, our analysis of Ser251 phosphorylation status in the context of linker domain phosphorylation site mutants revealed that the double mutant of Ser908 and Thr911 does have an effect on Ser251 phosphorylation status (Fig. 3c); the mutation of Ser908 and Thr911 to alanine results in a 60% reduction in Ser251 phosphorylation. Login to comment
348 This result is consistent with the fact that a triple mutant of Ser251Ala, Ser908Ala, and Thr911Ala has a phenotype identical to the Ser908Ala,Thr911Ala double mutant (Paumi et al., 2008), and supports the previous finding that the phenotype resulting from the Ser908Ala, Thr911Ala double mutant is dominant over the increased growth phenotype associated with the Ser251Ala mutant. Login to comment
352 Ycf1p-Ser872Ala and Ycf1p-Ser903Ala mutants also show decreased activity both in vivo and in vitro (Fig. 2a and d) similar to the Ycf1p-Ser908Ala, Thr911Ala mutant. Login to comment

[hide] Negative regulation of the yeast ABC transporter Y... J Biol Chem. 2008 Oct 3;283(40):27079-88. Epub 2008 Jul 29. Paumi CM, Chuk M, Chevelev I, Stagljar I, Michaelis S
Negative regulation of the yeast ABC transporter Ycf1p by phosphorylation within its N-terminal extension.
J Biol Chem. 2008 Oct 3;283(40):27079-88. Epub 2008 Jul 29., [PMID:18667437]

Abstract [show]
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.

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81 DH5␣ 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. Login to comment
88 Lastly, the TABLE 1 Yeast strains used in this study Straina Relevant genotype Reference SM4460b MATa ⌬met 15 ⌬leu2 ⌬ura3 ⌬his3 Mason et al. (23) SM5270 ⌬ycf1::KanMX Paumi et al. (34) SM5280 SM5270 with pSM1753 ͓2␮ YCF1-GFP URA3͔ Paumi et al. (34) SM5506 SM5270 with pSM2243 ͓2␮ YCF1-S251A-GFP URA3͔ This study SM5507 SM5270 with pSM2244 ͓2␮ YCF1-S251E-GFP URA3͔ This study SM5508 SM5270 with pSM2245 ͓2␮ YCF1-S908A,T911A-GFP URA3͔ This study SM5509 ⌬hal5::KanMX Open Biosystems SM5510 ⌬cka1::KanMX Open Biosystems SM5511 SM4460 with pSM1774 ͓2␮ YCF1-HAloop-GFP URA3͔c This study SM5512 SM4460 with pSM2246 ͓2␮ YCF1-S251A-HAloop-GFP URA3͔c This study SM5513 SM5509 with pSM1774 ͓2␮ YCF1-HAloop-GFP URA3͔c This study SM5514 SM5510 with pSM1774 ͓2␮ YCF1-HAloop-GFP URA3͔c This study SM5515 ⌬hal5::KanMX ⌬ycf1::URA3 This study SM5516 ⌬cka1::KanMX ⌬ycf1::URA3 This study SM5519 SM5510 with pSM1753 ͓2␮ YCF1-GFP URA3͔ This study SM5520 SM5510 with pSM2243 ͓2␮ YCF1-S251A-GFP URA3͔ This study SM5521 SM5510 with pSM2244 ͓2␮ YCF1-S251E-GFP URA3͔ This study SM5522 SM5509 with pSM1753 ͓2␮ YCF1-GFP URA3͔ This study SM5523 SM5509 with pSM2243 ͓2␮ YCF1-S251A-GFP URA3͔ This study SM5524 SM5510 with pSM2244 ͓2␮ YCF1-S251E-GFP URA3͔ This study SM5546 SM5270 with pSM2247 ͓2␮ YCF1-S251A,S908A,T911A-GFP URA3͔ This study L40 MATa, trp1, leu2, his3, LYS2::lexA-HIS3, URA3::lexA-lacZ Paumi et al. (34) THY AP4 MATa, ura3, leu2,lexA::lacZ::trp1, lexA::HIS3, lexA::ADE2 Paumi et al. (34) AP4-YCF1-CT THY.AP4 with YCF1::CT-KanMX Paumi et al. (34) AP4-SHO1-CT THY-AP4 with SHO1::CT-KanMX Paumi et al. (34) a All SM strains listed here are isogenic to SM4460. Login to comment
91 TABLE 2 Plasmid used in this study Plasmid Relevant genotype Reference pSM1753 ͓2␮ YCF1-GFP URA3͔ Mason et al. (23) pSM1774a ͓2␮ YCF1-HALoop-GFP URA3͔ Mason et al. (23) pSM2243 ͓2␮ YCF1-S251A-GFP URA3͔ This study pSM2244 ͓2␮ YCF1-S251E-GFP URA3͔ This study pSM2245 ͓2␮ YCF1-S908A,T911A-GFP URA3͔ This study pSM2246 ͓2␮ YCF1-S251A-HAloop-GFP URA3͔a This study pSM2306 ͓2␮ YCF1-S251A, S908A,T911A-GFP URA3͔ This study pPR3-N Contains the CYC1 promoter followed by the multiple cloning site and a hemagglutinin epitope. Login to comment
115 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). Login to comment
124 C-terminally GFP-tagged versions of these mutants were expressed in a ⌬ycf1 strain background along with WT Ycf1p and the previously described phosphorylation mutant Ycf1p-S908A,T911A (35). Login to comment
126 The somewhat diminished expression of Ycf1p-S908A,T911A seen here (ϳ50% of WT; Fig. 1B, compare lanes 2 and 5) can also be inferred from a study published by others (35). Login to comment
127 A slight mobility shift is also evident for the Ycf1p-S908A,T911A mutant protein, as was Negative Regulation of Ycf1p 27082 observed previously. Login to comment
131 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 ⌬ycf1 strain (row 2). Login to comment
133 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). Login to comment
156 In comparison, vacuoles derived from a Ycf1p-S908A,T911A strain have decreased transport activity, in agreement with what has been reported previously (35). Login to comment
160 Ycf1p-S908A,T911A kinetics have not been reported previously. Login to comment
162 The decrease in transport efficiency for Ycf1p-S908A,T911A is the result of both an increase in Kmapp for the substrate (E2beta17G) and a decrease in Vmax,app as compared with WT (Fig. 3B and Table 3). Login to comment
175 of E 2 β17G(µM ) Ycf1p-S251A Ycf1p-S251E Ycf1p Ycf1p-S908A,T911A 0 50 100 150 200 250[H3]E2β17G(nmole/10min/mg)A. B. FIGURE 3. Login to comment
178 Experiments were conducted in triplicate or quintuplet, and error bars represent mean Ϯ S.D. B, Michaelis-Menten kinetics of Ycf1p-dependent transport of [3 H]E2beta17G 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." Login to comment
181 TABLE 3 Michaelis-Menten kinetics for Ycf1p transport of ͓3 H͔E2beta17G Vacuole type Km,app a Vmax a Vmax/Km,app Relative efficiencyb ␮M nmol/10 min/mg Ycf1p 394 260 0.66 1.0 Ycf1p-S251A 187 246 1.32 2.0 Ycf1p-S251E 481 276 0.57 0.86 Ycf1p-S908A,T911A 548 154 0.28 0.42 a Values are extrapolated from Fig. 3B. Login to comment
231 In contrast, we observed an increased Km and decreased Vmax of transport for the S908A,T911A double mutant, suggesting that phosphorylation within the ABC core could negatively affect both substrate binding and transport. Login to comment