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. 2010 Jul 9;22(7):2353–2369. doi: 10.1105/tpc.110.073973

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© 2010 American Society of Plant Biologists

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Figure 6. — The WDxR Motif Is Essential for CSAat1A and B Interaction with DDB1A.

(A) Positions of single mutations generated in the WDxR motif. The numbers indicate the amino acid residue in CSAat1A and B. All indicated sites were mutated to Ala (A).

(B) and (C) Effect of single amino acid mutations in the WDxR motif on the interaction between CSAat1A or B and DDB1A in yeast. CSA1at1A or B, CSAat1A^D212A or B^D212A, CSAat1A^W218A or B^W218A, CSAat1A^D219A or B^D219A, CSAat1A^R221A or B^R221A, CSAat1A^L206A or B^L206A, CSAat1A^T208A, or B^T208A and CSAat1A^R216A or B^R216A in pGADT7 were cotransformed with pGBKT7-DDB1A into yeast. Growth of the transformed yeast was assayed on media minus Trp and Leu (left panels) or minus Trp, Leu, and His with 25 mM 3-amino-1,2,4-triazole (right panels). Columns in each panel represent serial decimal dilutions. BD, binding domain.

(D) and (E) Effect of single amino acid mutations in the WDxR motif on the interaction between CSAat1A or B and DDB1A-CUL4 in planta. The CSAat1A- or B-Flag proteins were immunoprecipitated with anti-Flag agarose. The pull-down products were analyzed via immunoblots with anti-Flag, anti-DDB1A, or anti-CUL4 antibodies. WB, immunoblot.

(F) and (G) Effect of multiple amino acid mutations in the WDxR motif on the interaction between CSAat1A or B and DDB1A in yeast. CSA1at1A or CSA1at1B (B); D1, CSAat1A^D212AW218A or B^D212AW218A; D2, CSAat1A^D212AD219A or B^D212AD219A; D3, CSAat1A^D212AR221A or B^D212AR221A; D4, CSAat1A^L206AD212A or B^L206AD212A; T1, CSAat1A^{D212AW218AD219A} or B^{D212AW218AD219A}; T2, CSAat1A^{W218AD219AR221A} or B^{W218AD219AR221A}; and Q, CSAat1A ^{D212AW218AD219AR221A} or B ^{D212AW218AD219AR221A} in pGADT7 were cotransformed with pGBKT7-DDB1A into yeast. Growth of the transformed yeast was assayed on media minus Trp and Leu (left panels) or minus Trp, Leu, and His with 25 mM 3-amino-1, 2,4-triazole (right panels). Columns in each panel represent serial decimal dilutions. BD, vector pGBKT7.

(H) and (I) Effect of multiple amino acid mutations in the WDxR motif on the interaction between CSAat1A or B and DDB1A-CUL4 in planta. The CSAat1A- or B-Flag proteins were immunoprecipitated with anti-Flag agarose. The pull-down products were analyzed via immunoblot analysis with anti-Flag, anti-DDB1A, or anti-CUL4 antibodies. W, wild type; 1A and 1B, 35Spro-CSAat1A- or B-Flag, respectively; D1, 35S-pro-CSAat1A^D212AW218A- or B^D212AW218A-Flag; D2, 35Spro-CSAat1A^D212AD219A- or B^D212AD219A-Flag; D3, 35Spro-CSAat1A^D212AR221A- or B^D212AR221A-Flag; D4, 35Spro-CSAat1A^D212AL206A- or B^D212AL206A-Flag; T1, 35Spro-CSAat1A^{D212AW218AD219A}- or B^{D212AW218AD219A}-Flag; T2, 35Spro-CSAat1A^{W218AD219AR221A}- or B^{W218AD219AR221A}-Flag; Q, 35Spro-CSAat1A^{D212AW218AD219AR221A}- or B^{D212AW218AD219AR221A}-Flag. WB, immunoblot.