1. Dynamic Control of Yeast MAP Kinase Network by Induced Association and Dissociation between the Ste50 Scaffold and the Opy2 Membrane Anchor 
Katsuyoshi Yamamoto, Kazuo Tatebayashi, Keiichiro Tanaka, Haruo Saito 
Molecular Cell 
Volume 40, Issue 1, Pages (October 2010)
DOI: /j.molcel
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2. Molecular Cell 2010 40, 87-98DOI: (10.1016/j.molcel.2010.09.011)
Copyright © 2010 Elsevier Inc. Terms and Conditions

3. Figure 1 Functional Domains in the Opy2 Cytoplasmic Region
(A) Schematic diagrams of Opy2 constructs. Numbers are amino acid positions. FL, full-length; SR, Ser-rich regions; TM, transmembrane segment; L, linker region; CR, conserved regions.
(B and C) Osmostress-induced HOG reporter expression. KY477 (ssk2/22Δ opy2Δ) (B) or KY517 (ssk2/22Δ opy2Δ STE11-Q301P) (C) was cotransformed with pRS414-8xCRE-lacZ and pRS416-Opy2 (Opy2-FL) or its deletion derivatives. Cells were grown in CAD and treated with (+) or without (−) 0.4 M NaCl for 30 min. The activity of β-galactosidase in cell extracts was normalized with cell densities and is expressed as Miller units (Miller, 1972). All reporter assays were carried out in triplicate (or more) with independent cultures. Error bars represent standard deviations (SDs).
(D) KY477 (ssk2/22Δ opy2Δ) cells containing the indicated Opy2 constructs were serially diluted (10-fold) and spotted on YPD plates containing the indicated concentrations of sorbitol and were grown at 30°C.
(E) TM257 (ssk2/22Δ) was cotransformed with p416TEF2-GST-Ste50∗ (Ste50∗: Ste A) and p414GAL1-Opy2ΔSR1-GFP (or its derivatives), grown in CARaf, incubated with 2% galactose for 1 hr, and further incubated in CARaf + 2% glucose for 30 min before cell extracts were prepared. GST-Ste50∗ (or GST) was precipitated from the cell extract with glutathione Sepharose, and coprecipitated Opy2-GFP was probed with an anti-GFP antibody. Circles indicate the positions of uncleaved Opy2ΔSR1-GFP proteins.
See also Figures S1 and S2.
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4. Figure 2 Ste50 Binding Sites in the Opy2 Cytoplasmic Region
(A) TM257 (ssk2/22Δ) was cotransformed with p414GAL1-Opy2ΔSR1-GFP (or its derivative) and p416TEF2-GST-Ste50∗. Cells were grown in CARaf, and the expression of Opy2ΔSR1-GFP was induced with 2% galactose for 1 hr. Cells were then transferred to CARaf + 2% glucose and lysed at the indicated time after transfer. GST-Ste50∗ was precipitated by glutathione Sepharose, and coprecipitated Opy2-GFP was probed by immunoblotting. The open triangle indicates the position of phosphorylated Opy2. Lys, total lysate (1:200 dilution); Glu, glucose.
(B) GST immunoprecipitate (IP: GST) similar to lane 4 in (A) was treated with λ protein phosphatase (λPPase) in the presence (+) or absence (−) of a phosphatase inhibitor mixture and probed by immunoblotting for coprecipitated Opy2ΔSR1-GFP.
(C) Same as in (A) except that the indicated Opy2 mutants were used. P-Opy2, phosphorylated Opy2.
(D) WT and mutant sequences of the CR-A, CR-B, and CR-D regions are shown. The numbers represent amino acid positions in the full-length Opy2 sequence. −, same as in the WT.
(E–H) KY477 (ssk2/22Δ opy2Δ) was transformed with pRS416-Opy2 (Opy2-FL) or its mutant derivatives. In (E), osmostress-induced 8xCRE-lacZ expression was assayed as in Figure 1B. Error bars represent SDs. In (F), cells were treated with 0.4 M NaCl for the indicated times. Total Hog1 (Hog1) and phosho-Hog1 (P-Hog1) levels were detected by immunoblotting of total cell extracts. In (G) and (H), the osmosensitivity of opy2 mutants was examined as in Figure 1D.
See also Figures S3 and S4.
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5. Figure 3 Glucose-Induced Phosphorylation of Opy2 CR-B
(A) Opy2 gel mobility shift assays were done as in Figure 2A, except that the indicated derivatives of p414GAL1-Opy2ΔSR1-GFP were used.
(B) TM257 was cotransformed with p424-Opy2ΔSR1-GFP and p416TEF2-GST-Ste50∗. Cells were grown in CAD (Glu). An aliquot was transferred to CAGly (Gly) and incubated for 1 hr before preparation of the cell extracts. GST-Ste50∗ was precipitated with glutathione Sepharose, and coprecipitated Opy2-GFP was probed by immunoblotting.
(C) KY477 (ssk2/22Δ opy2Δ) was transformed with either pRS414-Opy2 (WT) or its A∗ derivative, grown in CAD, washed once, and then transferred to CAD or to CAGly. After 1 hr incubation, osmostress-induced Hog1 phosphorylation was detected as in Figure 2F.
(D and E) Opy2 gel mobility shift assays. In (D), TM257 was triply transformed with p414GAL1-Opy2ΔSR1-GFP (or its B∗ derivative), p416TEF2-GST-Ste50∗, and either p423GAL1-HA-Yck1 (+) or the empty vector (−). Cells were grown in SRaf, and the expression of Opy2ΔSR1-GFP and HA-Yck1 was induced by 2% galactose for 1 hr before preparation of cell extracts. In (E), LRB939 (WT) and LRB1613 (yck1Δ yck2-ts) were cotransformed with p414GAL1-Opy2ΔSR1ΔC-GFP and p416TEF2-GST-Ste50∗ and were grown at 24°C. Opy2 expression was induced with 2% galactose for 45 min at 24°C and 15 min at 37°C, followed by the addition of 2% glucose. Cells were further incubated at 37°C for the indicated times before preparation of the cell lysates.
(F) KY531 (ssk2/22Δ opy2Δ YCK1 YCK2) and KY559 (ssk2/22Δ opy2Δ yck1Δ yck2-ts) were cotransformed with single-copy plasmids for 8xCRE-lacZ and Opy2 (or its indicated derivatives) and were grown in SC at 24°C. A portion of the culture was transferred to 37°C for 1 hr. Reporter expression was induced by 0.4 M NaCl for 30 min and was assayed as in Figure 1B. Error bars represent SDs.
Molecular Cell  , 87-98DOI: ( /j.molcel )
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6. Figure 4 Distinct Roles of CR-A and CR-B
(A) TM257 (ssk2/22Δ SNF3 RGT2) and KT174 (ssk2/22Δ snf3Δ rgt2Δ) were cotransformed with p424-Opy2ΔSR1-GFP and p416TEF2-GST-Ste50∗ and were grown in CAD to prepare cell extracts for gel mobility shift assay.
(B) Σ strains F1950 (WT) and KY550 (opy2Δ) were transformed with pRS414-Opy2 (WT), its mutant derivatives, or the empty vector (vec). Overnight culture (5 μl) was spotted on an YPD agar plate, incubated for 6 days at 30°C, and washed gently under running water.
(C–F) Crosstalk induction of FUS1-lacZ expression. KT077 (ssk2/22Δ opy2Δ pbs2Δ) and its indicated derivatives were cotransformed with pFUS1-lacZ and pRS414-Opy2 (or its indicated derivatives). Cells grown in CAD were treated with (blue) or without (white) 0.4 M NaCl for 3 hr before preparation of cell extracts for β-galactosidase assays. Error bars represent SDs.
(G and H) A working model for the regulation of the HOG and the FG/IG pathways by glucose signal.
See also Figure S5.
Molecular Cell  , 87-98DOI: ( /j.molcel )
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7. Figure 5
Phosphorylation of Ste50 by Activated MAPKs Decreases Ste50-Opy2 Interaction
(A) In vivo Ste50-Opy2 binding assay. TM257 (ssk2/22Δ) was cotransformed with p424-Opy2ΔSR1-GFP, p416TEF2-GST-Ste50 (or empty vector), and p413GALS-Ste11. The cells were grown in SC and were treated with 0.4 M NaCl as indicated before preparation of cell extracts. GST-Ste50 was precipitated, and associated Opy2-GFP was detected by immunoblotting.
(B) Ste50 gel mobility shift assays. FP67 (ssk2/22Δ ste50Δ) was co-transformed with YCpIF16-HA-Ste50 and either p416GALS-Ste11 (WT) or its kinase-dead K444A (K/A) derivative, was induced with 2% galactose for 2 hr, and was then treated with NaCl at the indicated concentration for 15 min. Cell extracts were separated by 7.5% SDS-PAGE, and HA-Ste50 was detected by immunoblotting.
(C) Cell extracts were prepared as in (B), except that the cells were stimulated with 0.4 M NaCl for 0 or 20 min and were treated with alkaline phosphatase (APase) in the presence or absence of APase inhibitors.
(D and E) The parental strains, TM257 (ssk2/22Δ) (D, lane 1), TM141 (SSK2+SSK22+) (E, lane 1), and QG158 (ssk2/22Δ) (E, lane 5) and their derivatives were cotransformed with YCpIF16-HA-Ste50 and p416GALS-Ste11. The Ste50 band shift was assayed as in (B), except that the cells were stimulated with 0.4 M NaCl for 20 min.
(F) Schematic diagram of Ste50 showing the locations of the MAPK phosphorylation motifs (SP or TP). The seven potential phosphorylation sites are numbered 1 through 7.
(G) FP67 was cotransformed with YCpIF16-HA-Ste50 or its mutant derivatives and p416GALS-Ste11. The Ste50 band shift was assayed as in (B), except that the cells were stimulated with 0.4 M NaCl as indicated.
(H) KY533 (FUS3 KSS1 bar1Δ) and KY535 (fus3Δ kss1Δ bar1Δ) were cotransformed with p416GALS-Ste11 and either YCpIF16-HA-Ste50 (WT) or its 23456A derivative (2-6A). Cells were stimulated with either 300 nM α factor (αF) for 44 min or 0.4 M NaCl for 20 min, as indicated. The Ste50 band shift was assayed as in (B).
(I) An in vivo Ste50-Opy2 binding assay was conducted as in (A), except that p416TEF2-GST-Ste A (GST-Ste50∗) was used in place of GST-Ste50.
See also Figure S6.
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8. Figure 6 Phosphorylated Ste50 Cannot Bind Opy2 In Vitro
(A) In vitro binding assay. Lysates were prepared as described in the Experimental Procedures. After the in vitro binding reaction, HA-Ste50 was immunoprecipitated, and coprecipitated Opy2-GFP was detected by blotting. In the right panel, Opy2-GFP in lysate 1 was probed. Open triangles indicate the positions of the Opy2-GFP constructs. U, unphosphorylated Ste50; P, phosphorylated Ste50.
(B) An in vitro binding assay was conducted as in (A), except that lysate 2 was prepared from cells that expressed either HA-Ste50 (WT) or HA-Ste A, together with either kinase-dead Ste11-K444A (K/A) or constitutively active Ste11-Q301P (Q/P), as indicated.
(C) An in vitro binding assay was conducted as in (A), except that lysate 1 contained GST-Ste11ΔC instead of Opy2-GFP. HA-Ste50 was immunoprecipitated, and coprecipitated GST-Ste11ΔC was detected by immunoblotting.
(D and E) Osmostress-induced Hog1 phosphorylation was detected as in Figure 2F. In (D), QG158 (ssk2/22Δ) and KY528 (ssk2/22Δ STE A) were grown in YPD. In (E), TA062 (ssk2/22Δ ste50Δ ptp2Δ) was transformed with either pRS414-Ste50 or pRS414-Ste A and grown in CAD.
See also Figure S7.
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9. Figure 7 Ste50 Phosphorylation Downregulates Multiple MAPK Pathways
(A and B) Cells were streaked on CAD (+ Glu) and CAGal (+ Gal) plates. In (A), strains of the indicated genotypes were transformed with pGAL1-Hkr1ΔSTR. In (B), KT048 (ste50Δ ssk2/22Δ) was cotransformed with pGAL1-Hkr1ΔSTR or pGAL1-Msb2ΔSTR and with pRS414-Ste50 (WT) or pRS414-Ste A.
(C) Congenic strains KY533 (Parental, ssk2/22Δ bar1Δ), KY534 (STE A), and KY535 (fus3Δ kss1Δ) were treated with (+) or without (−) 300 nM α factor for 44 min, followed by 6 min treatment with 0.4 M NaCl as indicated. Phosphorylated Hog1 and Fus3 were detected by immunoblotting.
(D and E) Crosstalk induction of FUS1-lacZ expression. The yeast strains TA064 (hog1Δ), TA066 (HOG1), KT175 (HOG1 ste4Δ), and FP55 (HOG1 ste5Δ) in (D), and TA066 (Parental), TA067 (msg5Δ), TA070 (ptp3Δ), and TA071 (msg5Δ ptp3Δ) in (E) were each cotransformed with pFUS1-lacZ and either p414-Ste50 (WT) or p414-Ste A (2-6A). Cells were grown in CAD and treated with or without 0.4 M NaCl for 3 hr before preparation of cell extracts for β-galactosidase assays. Error bars represent SDs.
(F) A schematic model of MAPK downregulation by phosphorylation of Ste50.
Molecular Cell  , 87-98DOI: ( /j.molcel )
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