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).

Myo4p globular tail is required for efficient ASH1 mRNA and Myo4p localization. (A) Representative images of mitotic yeast cells stained for ASH1 mRNA localization by fluorescent in situ hybridization (FISH, left images) and for nuclei by DAPI (right). (B) Graphical representation of the ASH1 mRNA distribution. Error bars represent the SD from three independent experiments of n = 3 × ≥70 cells. The observed differences in bud-tip localization are statistically significant (two-sided t test; significance level P = 0.05). (C) Representative images of yeast cells with mid-sized buds that were stained for Myo4p-myc9 (left). Images on the right show DAPI staining. (D) Graphical representation of the Myo4p distribution. Error bars represent the SD from three independent experiments of n = 3 × ≥250 cells. Bar (A and C): 4 µm.

Structural conservation of the globular tail of Myo4p. (A) Residues involved in auto-inhibition of type V myosins by their globular tail (Li et al., 2008) 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.

Asymmetric distribution of Ash1 protein. (A) A white/red assay was used to assess localized ASH1-mRNA translation. White color indicates correct asymmetric distribution of Ash1p, whereas a red color indicates defects in this process. As expected, Myo4p knock-out cells show a red color when grown on adenine-deficient media whereas wild-type Myo4p-expressing cells are white. myo4ΔGT cells show a red color. (B) On media containing 0.03% canavanine, myo4Δ and myo4ΔGT cells grow identically, whereas MYO4 cells show an impaired growth. Both experiments suggest the requirement of the globular tail domain for localized ASH1-mRNA translation at the tip of the daughter cell. (C and D) Assessment of Ash1p expression levels in wild-type and myo4ΔGT cells by quantitative Western blot. (C) Exemplary Western blot with each sample loaded three times for accurate quantification of Ash1p-Myc and Pgk1p expression levels. (D) The relative expression of Ash1p showed no considerable difference between wild-type and myo4ΔGT cells. We normalized the expression of Ash1p relatively to Pgk1p by setting their ratio to one in Myo4p wild-type cells. Three independent experiments were performed for each strain.

Crystal structure of the Myo4p globular tail. (A) Cartoon representation of the Myo4p globular tail structure, colored in rainbow representation with the N terminus in blue and C terminus in red. The N and C termini are located next to each other. (B) The Myo4p globular tail, rotated by 180° around the x axis. Subdomain I is highlighted in blue and sub-domain II in red. (C) Topogram of the Myo4p globular tail structure. Colors are the same as in B, except for the loop-connecting helices H13 and H14, which are represented in gray. (D) Surface charge representation of the Myo4p globular tail, with positively and negatively charged areas shown in blue and red, respectively. Cartoon representations were generated with PyMOL (DeLano Scientific LLC), surface charges with CCP4mg (Potterton et al., 2002).

Point mutants in the globular tail and the linker region of Myo4p impair She3p binding and ASH1 mRNA localization. (A) Cartoon representation of Myo4p fragments containing point mutations. Representation is identical to Fig. 1 A. (B) Ni-sepharose pull-down reactions with immobilized His-She3p-N and point-mutated Myo4p fragments. Whereas both Myo4p fragments with double point mutations in their globular tail showed no detectable interaction (right), the same Myo4p fragment without point mutations yielded significant binding (left). (C) Graphical representation of the ASH1 mRNA localization in myo4Δ cells, rescued by the expression of either wild-type Myo4p or its mutant forms Myo4p-(W1325D,Y1329D) and Myo4p-(F1056R,I1057R). ASH1 mRNA localization is the same as in Fig. 3, A and B. Both mutants show statistically significant defects in bud-tip localization (two sided t-test; significance level P = 0.05). SDs are derived from three independent experiments of n = 3 × ≥50 cells (total >200 cells per construct).