Two-hybrid interactions between She4p and Myo3/5p. DNA fragments encoding various regions of She4p and Myo3/5p were cloned into pBTM (LexA DNA-binding domain plasmids) and pGAD (GAL4 transcriptional activating domain plasmids) vectors, respectively. The resultant plasmids were introduced into L40 cells. The interactions between She4p and Myo3/5p were examined qualitatively by histidine auxotrophy and quantitatively by β-galactosidase activity assay. The numbers in parentheses represent amino acid positions. Closed bars and open bars stand for His+ and His-, respectively. β-galactosidase (LacZ) activity represents the average and SD for two independent transformants. (A) Interactions between She4p (full length) and various regions of Myo3/5p. ATP, putative ATP-binding domain; IQ, IQ motifs; SH3, Src homology 3 domain. (B) Interactions between various regions of She4p and Myo3p (471–572).

Effects of SHE4 disruption or overexpression on the two-hybrid interactions between Myo3p motor and Act1p. (A) L40 (wild-type) and YKT550 (she4Δ) cells were transformed with pKT1006 (pDBD-MYO3 motor) and either pACTII-HK-ACT1 (pAD-ACT1) or pACTII-HK (pAD-Vector), and the interaction between the motor domain of Myo3p and Act1p was examined qualitatively by histidine auxotrophy. (B) TAT7 cells containing pKT1006 were transformed simultaneously with pACTII-HK-ACT1 and either pKT1294 (multicopy SHE4) or YEplac195 (vector). The interaction between the motor domain of Myo3p and Act1p was examined quantitatively by β-galactosidase activity assay. Bar graph shows the average and SD for five independent transformants.

Novel dominant point mutations in the MYO5 motor region can bypass the requirement of She4p. (A) Effects on the temperature-sensitive growth defect of she4Δ cells. Plasmids pKT1332 (V164I), pKT1333 (N168I), pKT1334 (N209S), pKT1335 (K377M), pKT1330 (wild-type), and YEplac195 (vector) were introduced into YKT275 cells. Transformants were selected on SDA-Ura plates and streaked on YPDA plates. The plates were incubated for 2 d at 25°Cor37°C. (B) Three-dimensional structure of chicken skeletal muscle myosin S1 (the Protein Data Bank code, 2MYS). Residues corresponding to V164, N168, N209, and K377 of the yeast Myo5p (I222, N226, D264, and K431 of chicken skeletal muscle myosin S1, respectively; Cope and Hodge, 2000) are represented in space-filling format. These residues are located inside the cleft, which extends from the ATP-binding pocket to the actin-binding site and separate the upper and the lower 50-kDa subdomains. This picture was generated using RasMol (Sayle and Milner-White, 1995). (C) Effects on polarized localization of cortical actin patches in she4Δ cells. Plasmids YEplac195 (vector), pKT1330 [MYO5 (WT)], pKT1332 (V164I), pKT1333 (N168I), pKT1334 (N209S), pKT1335 (K377M), and pKT1290 (SHE4) were introduced into YKT275 cells. Transformants were grown in SDA-Ura liquid media at 25°C, fixed, and stained with TRITC-phalloidin. Top panels show localization of F-actin of she4Δ cells with plasmid pKT1332 [MYO5 (V164I)] or pKT1290 (SHE4). Bar, 5 μm. The bar graph shows the percentage of polarized cell (no more than three patches in the mother cell; see MATERIALS AND METHODS). (D) Effects on fluid-phase endocytosis defect of she4Δ cells. pKT1334 (N209S), pKT1330 (WT), and YEplac195 (vector) were introduced into YKT275. Cells grown in SDA-Ura at 25°C were incubated with lucifer yellow at 25°C for an additional 30 min. Accumulation of lucifer yellow in the vacuole was visualized using an fluorescein isothiocyanate band pass filter (LY). The same exposure and processing parameters were used for comparison. Bar, 5 μm.

SHE4 overexpression exacerbates the temperature-sensitive growth defect of the myo5 motor mutant. (A) YKT91 (myo5-1), YKT93 (myo5-218), YKT111 (myo5-360), and YKT110 (MYO5) cells were transformed with pKT1294 (YEp-SHE4) or YEplac195 (vector). Transformants were streaked on SDA-Ura plates and incubated for 3 d at 30°C (myo5-218 and myo5-360) or 35°C (myo5-1 and MYO5). (B) The domain structure of Myo5p and the amino acid substitution(s) of myo5-1 (closed circle), myo5-218 (stars), and myo5-360 (triangles) are depicted. ATP, putative ATP-binding domain; IQ, IQ motifs; SH3, Src homology 3 domain.

She4p interacts with type II and type V myosins and is required for proper localization of Myo4p. (A) Two-hybrid interactions between She4p and myosins. DNA fragments encoding Myo1p, Myo2p, and Myo4p were cloned into pGAD vectors. The resultant plasmids were introduced into L40 cells carrying pKT1295. The interaction between the UCS domain of She4p and myosins were examined quantitatively by β-galactosidase activity assay. The myosin motor regions used in the experiment were represented schematically. The numbers below actin-binding surfaces and in parentheses represent amino acid positions in chicken skeletal muscle myosin and each yeast myosin, respectively. β-Galactosidase (LacZ) activity represents the average and SD for three independent transformants. (B) Localization of Myo1p-GFP in she4Δ cells. Myo1p-GFP–expressing cells of YKT640 (wild-type) and YKT641 (she4Δ) were exponentially grown at 25°C. Myo1p-GFP was visualized using a GFP band pass filter (Myo1p-GFP). The same exposure and processing parameters were used for comparison. Bar, 5 μm. (C) Localization of Myo2p-GFP in she4Δ cells. YKT520 (wild-type) and YKT524 (she4Δ) were observed as described in B. Bar, 5 μm. (D) Localization of Myo4p-GFP in she4Δ cells. YKT544 (wild-type) and YKT543 (she4Δ) were observed as described in B. Bar, 5 μm. (E) Delocalization of Myo4p-GFP in she4Δ mutant is not due to depolarization of the actin cytoskeleton. Myo4p-GFP–expressing cells, YKT543, were transformed with YEplac195 (vector), pKT1332 [MYO5 (V164I)], or pKT1290 (SHE4). Transformants were exponentially grown in SDA-Ura at 25°C, fixed, and stained with TRITC-phalloidin. Localization of Myo4p-GFP and F-actin was visualized with a GFP band pass filter (Myo4p-GFP) and a TRITC filter (actin), respectively. For each observation, the same exposure and processing parameters were used. Bar, 5 μm.

She4p is required for proper localization of Myo5p-GFP and F-actin. (A) Localization of Myo5p-GFP in she4Δ cells. Myo5p-GFP expressing cells, YKT159 (wild-type) and YKT288 (she4Δ) were grown at 25°C, fixed and stained with TRITC-phalloidin. Localization of Myo5p and F-actin was visualized with a GFP band pass filter (Myo5p-GFP) and a TRITC filter (actin). For each observation, the same exposure and processing parameters were used. Arrows indicate examples of colocalization of Myo5p-GFP and actin patches. (B) Localization of Myo5-1p-GFP. YKT114 cells were transformed with plasmids, pKT1235 (Myo5p-GFP) or pKT1403 (Myo5-1p-GFP). Cells were grown in SD-Leu liquid medium at 25°C, incubated at 37°C for 1 h, fixed, stained, and observed as described in A. (C) Localization of She4p-GFP. She4p-GFP–expressing cells, YKT274, were grown at 25°C, and She4p-GFP was visualized using a GFP band pass filter (She4p-GFP) or differential interference contrast optics (DIC). Bars, 5 μm.

Genetic interactions between SHE4 and actin-related genes and MYO5. (A) Summary of genetic interactions. Each mutant strain was crossed with she4Δ (YKT275 or YKT276), and resultant diploid cells were sporulated and dissected for tetrad analysis. The growth characteristics of the resulting double or triple mutants were determined at 25°C. The mutant strains used were YMW211U (arp2-1), YMW221U (arp2-2), YKT130 (vrp1Δ), YKT218 (sla1Δ), DDY322 (abp1Δ), DDY1520 (aip1Δ), YKT382 (bni1Δ), YKT388 (cla4Δ), DDY1226 (cof1-22), DDY1024 (pfy1-116), DDY949 (rvs167Δ), DDY318 (sac6Δ), YKT186 (sla2Δ), YKT91 (myo3Δ myo5-1), YKT93 (myo3Δ myo5-218), and YKT111 (myo3Δ myo5-360). (B) Genetic interactions of arp2-1, arp2-2, sla1Δ, and vrp1Δ with she4Δ mutation. Diploid cells obtained from an indicated cross were sporulated, dissected, and grown at 25°C for 3–4 d before being photographed. Colonies were then replica plated to determine the segregation of the marked mutant alleles. Tetrad genotype (TT, tetratype; PD, parental ditype; and NPD; nonparental ditype) is indicated, and the identity of the double mutant spore(s) is shown in parentheses.

She4p interacts with Myo5p in a temperature-dependent manner. Myo5p-TAP from YKT323 lysate was adsorbed to IgG Sepharose. GST-She4p was purified from bacterial lysate. IgG Sepharose with or without Myo5p-TAP was incubated with GST-She4p at 4 or 30°C. The IgG Sepharose was collected, and bound proteins were analyzed by SDS-PAGE and SYPRO orange staining. The result shown is a representative of four experiments.