Figure 2. Assembly of the SHE core... | Rockefeller University Press

$Figure 2. Assembly of the SHE core complex induces Myo4p dimerization. (A) Quantitative mass spectrometry of the SHE core complex reconstituted with ASH1-E3-51 or ASH1-E3-77-tRNA. Heavy isotope-labeled peptides of She3p and Myo4p-C were used for quantification. The stoichiometry of the She3p:Myo4p-C interaction was 2.12 (ASH1-E3-51 complex) or 2.23 (ASH1-E3-77-tRNA complex). (B) The stoichiometry of the SHE core complex was determined by EMSA with radioactively labeled RNA. A constant concentration of ASH1-E3-77:She2p (2:4) subcomplex was used, and increasing concentrations of She3p:Myo4p-C (2:1) subcomplex were titrated. The distinct band observed in lane 6 (double arrow and red box) indicates binding of two She3p:Myo4p subcomplexes to one RNA-bound She2p tetramer. The single arrow marks a substoichiometric SHE core complex, and the asterisk marks a high molecular mass complex between the RNA in the SHE core complex and excess She3p:Myo4p-C with unspecific RNA-binding properties. (C) SLS profile of the SHE core complex containing ASH1-E3-77-tRNA, She2p, She3p, and Myo4p-C. RI, refractive index; RALS, right angle light scattering; MM, fitted molecular mass.$

Figure 2.

Assembly of the SHE core complex induces Myo4p dimerization. (A) Quantitative mass spectrometry of the SHE core complex reconstituted with ASH1-E3-51 or ASH1-E3-77-tRNA. Heavy isotope-labeled peptides of She3p and Myo4p-C were used for quantification. The stoichiometry of the She3p:Myo4p-C interaction was 2.12 (ASH1-E3-51 complex) or 2.23 (ASH1-E3-77-tRNA complex). (B) The stoichiometry of the SHE core complex was determined by EMSA with radioactively labeled RNA. A constant concentration of ASH1-E3-77:She2p (2:4) subcomplex was used, and increasing concentrations of She3p:Myo4p-C (2:1) subcomplex were titrated. The distinct band observed in lane 6 (double arrow and red box) indicates binding of two She3p:Myo4p subcomplexes to one RNA-bound She2p tetramer. The single arrow marks a substoichiometric SHE core complex, and the asterisk marks a high molecular mass complex between the RNA in the SHE core complex and excess She3p:Myo4p-C with unspecific RNA-binding properties. (C) SLS profile of the SHE core complex containing ASH1-E3-77-tRNA, She2p, She3p, and Myo4p-C. RI, refractive index; RALS, right angle light scattering; MM, fitted molecular mass.

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