Analysis of acid phosphatase secretion and effect of Rho3 overproduction.

<p>Wild-type cells and Δ<i>apm1</i> cells, which were transformed with either the pDB248 vector or the <i>rho3⁺</i>-containing vector, were assayed for acid phosphatase activity as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016842#s2" target="_blank">Materials and Methods</a> section. Values from 3 independent experiments are expressed as means ± standard deviation.</p

Rho3 suppressed various phenotypes associated with Apm1-deletion cells.

<p>(A) Rho3 suppressed the defective localization of GFP-Syb1 in Δ<i>apm1</i> cells. Wild-type cells (wt) and Apm1-deletion (Δ<i>apm1</i>) expressing chromosome-bone GFP-Syb1 cells transformed with pDB248 or the vector containing <i>rho3⁺</i> was cultured in YPD medium at 27°C. The GFP-Syb1 localization was examined under the fluorescence microscope. Bar 10 µm. (B) Rho3 suppressed the defective localization of FM4-64 in Δ<i>apm1</i> cells. Wild-type (wt) and Apm1-deletion cells (Δ<i>apm1</i>) transformed with pDB248 or the vector containing <i>rho3⁺</i> were cultured in YPD medium at 27°C. Cells were collected, labeled with FM4-64 fluorescent dye for 5 min, resuspended in water, and examined by fluorescence microscopy. Bar 10 µm. (C) Wild-type (wt) and Apm1-deletion cells (Δ<i>apm1</i>) transformed with pDB248 or the vector containing <i>rho3⁺</i> cultured in YPD medium at 27°C. Cells were collected, labeled with FM4-64 fluorescent dye for 60 min, resuspended in water, and examined by fluorescence microscopy. Bar 10 µm. (D) Wild-type cells and Δ<i>apm1</i> cells, which were transformed with either the pDB248 vector or the <i>rho3⁺</i>-containing vector, were assayed for acid phosphatase activity as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016842#s2" target="_blank">Materials and Methods</a> section. Values from 3 independent experiments were plotted as means ± S.D.</p

Isolation of Rho3 as a multicopy suppressor of <i>apm1</i> mutant cells.

<p>(A) The <i>apm1</i> mutant cells (<i>apm1-1</i>) were transformed with either the pDB248 multicopy vector, the vector containing <i>apm1⁺</i> or the vector containing <i>rho3⁺</i>. Cells were then streaked onto plates containing YPD, YPD plus 0.2 M MgCl₂, YPD plus 0.5 µg/mL FK506, EMM, or EMM plus 0.5 µg/mL FK506 and then incubated for 4 d at 27°C or for 3 d at 36°C, respectively. (B) Cells transformed with the multicopy vector pDB248, or the genome DNA clones containing <i>rho1⁺</i>, <i>rho2⁺</i>, <i>rho3⁺</i>, <i>rho4⁺</i>, <i>rho5⁺</i>, or <i>cdc42⁺</i> were streaked onto plates containing YPD and incubated for 4 d at 27°C or 3 d at 35°C, respectively.</p

Functional and physical interaction between Rho3 and Apm1.

<p>(A) Rho3 suppressed Apm1-deletion cells (Δ<i>apm1</i>) in a GTP- and effector domain-dependent manner. Apm1-deletion cells (Δ<i>apm1</i>) were transformed with the pDB248 multi-copy vector or the vector containing <i>rho3⁺</i>, <i>rho3</i>GV, <i>rho3</i>TN, <i>and rho3</i>EV expressed from its endogenous promoter. Cells were spotted onto each YPD plate and then incubated for 3 d. Cells were spotted in serial 10-fold dilutions starting with OD₆₆₀ = 0.6 of log-phase cells (5 µL). (B) Rho3 binds to Apm1 in a GTP- and effector domain-dependent manner. GST pull-downs performed with GST alone or Apm1-GST; cells expressing GFP-Rho3, GFP-Rho3GV, GFP-Rho3TN, or GFP-Rho3EV were collected, and the lysates were incubated with purified full-length Apm1 fused GST or the control GST protein. Proteins bound to glutathione beads were analyzed by SDS-PAGE and visualized by autoradiography. (C) Influence of Apm1 on the electropherograms of GFP-Rho3 protein determined by affinity capillary electrophoresis. Capillary, carboxylated fused silica (80 cm, 40 cm, 50 µm i. d.); BGE, 50 mM phosphate buffer (pH 6.8) containing Apm1-GST (gray line) or GST-Rho3 (black line: control) at a concentration of 200 µg/mL; applied voltage, 15 kV; temperature, 25°C; detection, laser-induced fluorescence detector (Ex, 473 nm; Em, 500-600 nm); sample introduction, hydrostatic method (10 cm ×10 s). (D) Comparison of the migration time-shifts in various Rho3 proteins. Δt (migration time-shift) is equal to t_control - t_affinity, where t_control and t_affinity are the migration times of GFP-Rho3 or its mutant versions in the absence (control: GST-Rho3) or presence (affinity: Apm1-GST) of Apm1-GST, respectively. Left panel: The experiments were performed at a protein concentration of 200 µg/mL. Right panel: The experiments were performed at a protein concentration of 100 µg/mL.</p

Functional interaction of Rho3 with the clathrin adaptor-protein complex.

<p>(A) Rho3 suppressed the temperature-sensitive growth of deletion mutants of individual adaptin subunit of the AP-1 complex. The Δ<i>apl2</i>, Δ<i>apl4</i>, and Δ<i>aps1</i> cells were transformed with either the pDB248 multicopy vector or with the <i>rho3⁺</i>-containing vector. The cells were then streaked onto plates containing YPD, and then incubated at 27°C for 4 d or at 35°C for 3 d, respectively. (B) Rho3 suppressed the immunosuppressant sensitivity of deletion mutants of individual adaptin subunit of the AP-1 complex. Cells as indicated in (A) were spotted onto YPD or YPD plus 0.5 µg/mL FK506 and incubated at 27°C for 4 d. (C) Binding assay involving Rho3 and the 4 subunits of the AP-1 complex. GST pull-down experiments performed with GST alone or Apm1-GST, GST-Apl2, Apl4-GST or GST-Aps1; cells expressing GFP-Rho3 were collected, and the lysates were incubated with purified full-length Apm1 fused GST, GST-Apl2, Apl4-GST or GST-Aps1 or the control GST protein. GST and GST-tagged adaptin subunits were precipitated by glutathione beads, washed extensively, subjected to SDS-PAGE, and immunoblotted using anti-GFP or anti-GST antibodies.</p

<i>Schizosaccharomyces pombe</i> strains used in this study.

<p><i>Schizosaccharomyces pombe</i> strains used in this study.</p

GFP-Rho3 localizes at the Golgi/endosome.

<p>(A) The colocalization of GFP-Rho3 with FM4-64 in wild-type cells. Wild-type (wt) cells, expressing chromosome-bone GFP-Rho3 under the control of the <i>nmt1</i> promoter, were examined by fluorescence microscopy under the repressed conditions. The cells were incubated with FM4-64 fluorescent dye for 5 min at 27°C to visualize Golgi/endosomes. The fluorescence of the adaptin subunit and FM4-64 was examined under the fluorescence microscope. Arrows indicate the plasma membrane and medial region. Arrowheads indicate the dot-like structures and Golgi/endosomes. Bar 10 µm. (B) The partial colocalization of GFP-Rho3 with Apm1-mCherry in wild-type cells. Wild-type cells expressing chromosome-borne GFP-Rho3 were transformed with pREP1-Apm1-mCherry. The cells were cultured and observed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016842#pone-0016842-g006" target="_blank">Figure 6</a> (A). Bar: 10 µm.</p

Rho3-deletion cells displayed phenotypes similar to those seen in Apm1-deletion cells.

<p>(A) Wild-type (wt), Rho3-deletion (Δ<i>rho3</i>), and Apm1-deletion cells (Δ<i>apm1</i>) expressing chromosome-bone GFP-Syb1 cultured in YPD medium at 27°C were incubated with FM4-64 fluorescent dye for 5 min at 27°C to visualize Golgi/endosomes. GFP-Syb1 localization and FM4-64 fluorescence were examined under a fluorescence microscope. Bar 10 µm. (B) Wild-type (wt), Rho3-deletion (Δ<i>rho3</i>), and Apm1-deletion cells (Δ<i>apm1</i>) were cultured in YPD medium at 27°C. Cells were collected, labeled with FM4-64 fluorescent dye, resuspended in water, and examined by fluorescence microscopy. Photographs were taken after 60 min. Bar: 10 µm. (C) Wild-type (wt), Rho3-deletion (Δ<i>rho3</i>), and Apm1-deletion cells (Δ<i>apm1</i>) were streaked onto plates containing YPD or YPD plus 1.0 µg/mL micafungin, 0.5 µg/mL FK506, 0.3 M MgCl₂, and 6 mM valproic acid, and were then incubated at 27°C for 4 d or at 36°C for 3 d.</p