Journal: The Journal of Cell Biology

Article Title: The structure of the Myo4p globular tail and its function in ASH1 mRNA localization

doi: 10.1083/jcb.201002076

Figure Lengend Snippet: Efficient She3p binding to Myo4p requires the protease-sensitive linker and the globular tail. (A) Cartoon representation of Myo4p fragments. The full Myo4p tail consists of a coiled-coil region, a linker region, and a sequence stretch with 25% homology to the globular tail of Myo2p. Limited proteolysis experiments with the Myo4p tail fragment revealed a stable cleavage product of 45 kD ( Fig. S1 A ), which was identified as the globular tail. (B) Ni-sepharose pull-down reactions with immobilized His-She3p-N and different Myo4p constructs indicate that the protease-sensitive linker is required for efficient She3p-N binding. (C) Surface-plasmon resonance (SPR) with surface-coupled She3p-N reveals efficient binding of the Myo4p tail, whereas no binding was observed for the globular tail at concentrations up to 5 µM. (D) Pull-down reactions as in B with Myo4p tail in standard buffer (left), buffer containing additional 0.1% Triton X-100 (middle), or buffer containing 1 M NaCl (right). (E) Pull-down reaction as in D with a mutated Myo4p tail fragment (Myo4p-tail (F1056R, I1057R); see A). This fragment failed to reveal binding under standard conditions. (F) Pull-down reactions as in B with GST-Myo4p-L-GT (left), with a globular tail-lacking Myo4p fragment (GST-Myo4p-L; middle), or with a Myo4p fragment that has exchanged its globular tail with the corresponding domain of its paralogue Myo2p (GST-Myo4p-L-Myo2p-GT; right). Dots indicate the position of the respective Myo4p fragments. Note that in the input lane of the middle image, the top band is Myo4p, the middle band is She3p, and the bottom band is a degradation product of Myo4p. (G) SPR with surface-coupled She3p-N reveals efficient binding of the GST-Myo4p-L-GT, whereas no binding was observed even at ∼10 µM when the globular tail was exchanged for the corresponding domain of Myo2p (GST-Myo4p-L-Myo2p-GT).

Article Snippet: Because our attempts to obtain phase information by molecular replacement with the globular tail of Myo2p (RCSB Protein Databank ID: 2F6H ) as the template failed, we determined the structure by single-wavelength anomalous diffraction using crystals from selenomethionine-derivatized proteins ( ).

Techniques: Binding Assay, Sequencing, Construct, SPR Assay

Journal: The Journal of Cell Biology

Article Title: The structure of the Myo4p globular tail and its function in ASH1 mRNA localization

doi: 10.1083/jcb.201002076

Figure Lengend Snippet: Structural conservation of the globular tail of Myo4p. (A) Residues involved in auto-inhibition of type V myosins by their globular tail are conserved in Myo2p (green residues, left) but not in Myo4p (green residues, right), suggesting a lack of auto-inhibition in Myo4p. (B) Surface plot of a sequence alignment from Myo4p, Myo2p, and human Myo5a ( Fig. S2 ) show few regions with pronounced sequence identity. Dark yellow indicates residues identical in all three homologues, light yellow shows residues conserved in two homologues, and gray means no conservation. (C) Table showing that surface-exposed residues in Myo4p are much less conserved than buried residues, indicating a lack of conservation of potential binding surfaces.

Article Snippet: Because our attempts to obtain phase information by molecular replacement with the globular tail of Myo2p (RCSB Protein Databank ID: 2F6H ) as the template failed, we determined the structure by single-wavelength anomalous diffraction using crystals from selenomethionine-derivatized proteins ( ).

Techniques: Inhibition, Sequencing, Binding Assay

Journal: The Journal of Cell Biology

Article Title: The structure of the Myo4p globular tail and its function in ASH1 mRNA localization

doi: 10.1083/jcb.201002076

Figure Lengend Snippet: Plasmids

Article Snippet: Because our attempts to obtain phase information by molecular replacement with the globular tail of Myo2p (RCSB Protein Databank ID: 2F6H ) as the template failed, we determined the structure by single-wavelength anomalous diffraction using crystals from selenomethionine-derivatized proteins ( ).

Techniques: Expressing, Transformation Assay