Figure 1.
Isolation of Rho3 as a multicopy suppressor of sip1-i4 mutant cells.
(A) sip1-i4 cells were transformed with either the pDB248 multicopy vector, vector containing sip1+ or vector containing rho3+. Cells were then streaked onto plates containing 0.5 µg/mL FK506, 0.5 µg/mL micafungin, 0.3 M MgCl2, or 6 mM valproic acid and incubated at 27°C for 4 d or at 36°C for 3 d, respectively. (B) sip1-i4 cells transformed with the multicopy vector pDB248, or the genome DNA clones containing rho1+, rho2+, rho3+, rho4+, rho5+, or cdc42+ were streaked onto plates containing 0.5 µg/mL FK506, 0.3 M MgCl2, or 6 mM valproic acid then incubated at 27°C for 4 d or at 36°C for 3 d, respectively.
Figure 2.
Rho3 suppresses various phenotypes associated with sip1-i4 mutant cells.
(A) Rho3 suppresses the defective secretion of acid phosphatase in sip1-i4 mutant cells. Wild-type (wt) and sip1-i4 cells, which were transformed with either the pDB248 vector or rho3+-containing vector, were assayed for acid phosphatase activity. Data are representative of 3 independent experiments. (B) Rho3 suppresses the defects in vacuole fusion in sip1-i4 cells. The wt and sip1-i4 cells transformed with pDB248 or the vector containing rho3+ were cultured in YPD medium at 27°C. Cells were harvested, labeled with FM4-64 fluorescent dye for 60 min, resuspended in water, and examined by fluorescence microscopy. Bar, 10 µm. The number in the image indicates the percentage of cells with fragmented vacuoles. Data from at least 3 independent experiments are expressed as means ± standard deviations. (C) Rho3 suppresses GFP-Syb1 mislocalization in sip1-i4 mutant cells. The wt and sip1-i4 cells expressing GFP-Syb1 transformed with pDB248 or the vector containing rho3+ were cultured in YPD medium at 27°C. GFP-Syb1 localization was examined under a fluorescence microscope. Arrowheads indicate the dot-like structures of GFP-Syb1 and the Golgi/endosomes stained with FM4-64, double arrowheads indicate cytoplasmic accumulation, and arrows indicate the concentrated fluorescence at the medial region and cell surface. Bar, 10 µm. (D) Quantitative analysis of the number of Syb1 dots that co-localized with FM4-64/cell. (E) Percentage of cells in which Syb1 was localized at the cell surface. Cells in D and E were the same as those indicated in C.
Figure 3.
Functional and physical interactions between Rho3 and Sip1.
(A) Rho3 suppresses sip1-i4 mutant cells (sip1-i4) in a GTP- and effector domain-dependent manner. The sip1-i4 cells were transformed with the pDB248 multi-copy vector or the vector containing rho3+, rho3GV, rho3TN, and rho3EV expressed from its endogenous promoter. These cells were streaked onto YES plates and then incubated at 27°C for 4 d or at 36°C for 3 d, respectively. (B) Binding assay for Sip1 and Rho3. GST pull-down experiments were performed using chromosome-borne GST-Sip1 expressed under the control of the nmt1 promoter. Cells expressing GFP alone, or GFP-Rho3, GFP-Rho3GV, GFP-Rho3TN, or GFP-Rho3EV were harvested and their lysates were incubated with the purified full-length Sip1 fused GST protein. GST-tagged Sip1 was precipitated with glutathione beads, washed extensively, subjected to SDS-PAGE, immunoblotted using anti-GFP or anti-GST antibodies and visualized by autoradiography. Lower panel: Quantitation of GFP-tagged various mutant forms of Rho3 beads protein levels by densitometry of the expressed bands against that of the lysate protein levels as shown in B. Data from at least three independent experiments are expressed as means ± standard deviations.
Figure 4.
Both of Rho3 and Apm1 fail to co-localize at the Golgi/endosomes in sip1-i4 mutant cells.
(A) Subcellular localization of Apm1-GFP in wild-type (wt) and sip1-i4 mutant cells (sip1-i4). Cells expressing Apm1-GFP were cultured in YPD medium at 27°C, were incubated with the dye FM4-64 for 5 min at 27°C to visualize the Golgi/endosomes. The fluorescence of the FM4-64 was examined under the fluorescence microscope. Arrowheads indicated the localization of Apm1-GFP to the Golgi/endosomes. Bar, 10 µm. (B) Subcellular localization of GFP-Rho3 in wild-type (wt) and sip1-i4 mutant cells (sip1-i4). Cells expressing Rho3 were cultured in YPD medium at 27°C, following which they were incubated with FM4-64 dye for 5 min at 27°C to visualize the Golgi/endosomes. FM4-64 fluorescence was examined using a fluorescence microscope. Arrowheads indicate the dot-like structures of GFP-Rho3 and the Golgi/endosomes stained with FM4-64, double arrowheads indicate cytoplasmic accumulation, and arrows indicate the concentrated fluorescence at the cell division site. Bar, 10 µm. (C) Percentage of cells in which Rho3 were localized at the cell division site in wild-type (wt) and sip1-i4 cells. (D) Quantitative analysis for the number of Rho3 dots co-localizing with FM4-64/cells in wt and sip1-i4 cells. Cells in C and D were incubated as those in B. Data are the means ± standard deviations of 3 independent experiments with 150 cells in C and D.
Figure 5.
The Sip1 C-terminus is dispensable for the Sip1 association with the AP-1 complex or Rho3.
(A) Schematic representation of Sip1 protein, Sip1-i4 mutant protein and Sip1ΔN protein. The Sip1 protein is 1919 amino acids long and contains HEAT repeats (black). Star represents termination codon at the amino acid position 1434 found in the sip1-i4 allele. The Sip1-i4 mutant protein lacks the C-terminal 485 amino acids. The Sip1ΔN protein lacks the N-terminal 1414 amino acids. (B) Binding assay involving Sip1-i4 and the 4 subunits of the AP-1 complex. GST pull-down experiments were performed using Sip1-i4-GST, Sip1-i4-GST expressed under the control of the nmt1 promoter. Cells that expressed GFP alone or GFP-tagged to the 4 subunits of the AP-1 complex were harvested, and their lysates were incubated with the purified Sip1-i4 fused GST protein. GST-tagged proteins were analyzed as shown in Figure 3B. (C) Binding assay involving Sip1ΔN and the 4 subunits of the AP-1 complex. The binding assay was performed as described in B. (D) Binding assay involving Sip1-i4 and various mutant forms of Rho3. GST pull-down experiments were performed using Sip1-i4-GST expressed under the control of the nmt1 promoter. Cells that expressed GFP alone or various GFP-tagged mutant forms of Rho3 were harvested, and their lysates were incubated with the purified Sip1-i4-GST protein. GST-tagged Sip1-i4 was precipitated with glutathione beads, washed extensively, subjected to SDS-PAGE, and immunoblotted using anti-GFP or anti-GST antibodies. (E) Binding assay involving Sip1ΔN and various mutant forms of Rho3. The binding assay was performed as described in (D). Lower panel: Quantitation of GFP-tagged various mutant forms of Rho3 beads protein levels by densitometry of the expressed bands against that of the lysate protein levels as shown in D and E. Data from at least three independent experiments are expressed as means ± standard deviations.
Figure 6.
The C-terminus of Sip1 is important for its Golgi/endosomal localization.
(A) Co-localization of Sip1-GFP, Sip1-i4-GFP or Sip1ΔN-GFP with FM4-64 in wild-type cells. The wild-type (wt) cells that expressed chromosome-borne Sip1-GFP or wt cells transformed with pREP1-Sip1-i4-GFP or pREP1-Sip1ΔN-GFP were examined by fluorescence microscopy under repressed conditions. The cells were incubated with FM4-64 fluorescent dye for 5 min at 27°C to visualize the Golgi/endosomes. FM4-64 fluorescence was examined using a fluorescence microscope. Arrowheads indicate the dot-like structures and the Golgi/endosomes. Bar, 10 µm. (B) Sip1ΔN suppresses sip1-i4 cells similar to Sip1. The wt and sip1-i4 cells were transformed with the pDB248 multi-copy vector or the vector containing sip1+, sip1-i4, and sip1ΔN expressed under the control of the nmt1 promoter. Cells were streaked onto plates containing 0.5 µg/mL FK506 and then incubated at 27°C for 4 d or at 36°C for 3 d, respectively.
Figure 7.
Sip1 links Rho3 to AP-1 complex.
(A) Binding assay involving Apm1 and Rho3 in wild-type, or sip1-i4 cells harboring the control vector or Sip1ΔN. GST pull-down experiments were performed using Apm1-GST expressed in wild-type (wt) and sip1-i4 mutant (sip1-i4) cells, which were transformed with the pDB248 multi-copy vector or the vector containing sip1ΔN expressed under the control of the nmt1 promoter. Cells that expressed GFP alone or GFP-Rho3 were harvested, and their lysates were incubated with purified full-length Apm1 fused to GST. Proteins bound to glutathione beads were analyzed by SDS-PAGE and visualized by autoradiography. Right panel: Quantitation of GFP-Rho3 beads protein levels by densitometry of the expressed bands against that of the lysate protein levels in wild-type cells, sip1-i4 cells or sip1-i4 cells with Sip1ΔN expression as shown in A. Data from at least three independent experiments are expressed as means ± standard deviations. (B) Subcellular localization of GFP-Rho3 in wild-type cells, sip1-i4 cells or sip1-i4 cells with Sip1ΔN expression. GFP-Rho3 expressed in wild-type (wt) and sip1-i4 mutant (sip1-i4) cells, which were transformed with the pDB248 multi-copy vector or the vector containing sip1ΔN expressed under the control of the nmt1 promoter. Cells were cultured in YPD medium at 27°C, following which they were incubated with FM4-64 dye for 5 min at 27°C to visualize the Golgi/endosomes. FM4-64 fluorescence was examined using a fluorescence microscope. Arrowheads indicate the dot-like structures of GFP-Rho3 and the Golgi/endosomes stained with FM4-64, double arrowheads indicate cytoplasmic accumulation of GFP-Rho3, and arrows indicate the concentrated fluorescence at the cell division site. Bar, 10 µm. (C) Percentage of cells in which Rho3 were localized at the cell division site in wild-type (wt) and sip1-i4 cells, which were transformed with the pDB248 multi-copy vector or the vector containing sip1ΔN expressed under the control of the nmt1 promoter. (D) Quantitative analysis for the number of Rho3 dots co-localizing with FM4-64/cells in wt and sip1-i4 cells, which were transformed with the pDB248 multi-copy vector or the vector containing sip1ΔN expressed under the control of the nmt1 promoter. Data are the means ± standard deviations of 3 independent experiments with 150 cells in B.