ABCMdb

PMID: 18667437

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., [PubMed]

Sentences

No. Mutations Sentence Comment

81 ABCC2 p.Ser908Ala ABCC2 p.Ser908Ala 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 ABCC2 p.Ser908Ala ABCC2 p.Ser908Ala 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 ABCC2 p.Ser908Ala ABCC2 p.Ser908Ala 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 ABCC2 p.Ser908Ala 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 ABCC2 p.Ser908Ala 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 ABCC2 p.Ser908Ala 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 ABCC2 p.Ser908Ala 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 ABCC2 p.Ser908Ala 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 ABCC2 p.Ser908Ala 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 ABCC2 p.Ser908Ala 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 ABCC2 p.Ser908Ala Ycf1p-S908A,T911A kinetics have not been reported previously. Login to comment 162 ABCC2 p.Ser908Ala 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 ABCC2 p.Ser908Ala 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 ABCC2 p.Ser908Ala 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 ABCC2 p.Ser908Ala 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 ABCC2 p.Ser908Ala 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