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Lack of GTP-bound Rho1p in secretory vesicles of Saccharomyces cerevisiae.

Abe M, Qadota H, Hirata A, Ohya Y - J. Cell Biol. (2003)

Bottom Line: Rho1p, an essential Rho-type GTPase in Saccharomyces cerevisiae, activates its effectors in the GTP-bound form.Rom2p, the GDP/GTP exchange factor of Rho1p, is preferentially localized on the plasma membrane even when vesicular transport is blocked.Based on these results, we propose that Rho1p is kept inactive in intracellular secretory organelles, resulting in repression of the activity of the cell wall-synthesizing enzyme within cells.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, FSB-101, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan.

ABSTRACT
Rho1p, an essential Rho-type GTPase in Saccharomyces cerevisiae, activates its effectors in the GTP-bound form. Here, we show that Rho1p in secretory vesicles cannot activate 1,3-beta-glucan synthase, a cell wall synthesizing enzyme, during vesicular transport to the plasma membrane. Analyses with an antibody preferentially reacting with the GTP-bound form of Rho1p revealed that Rho1p remains in the inactive form in secretory vesicles. Rom2p, the GDP/GTP exchange factor of Rho1p, is preferentially localized on the plasma membrane even when vesicular transport is blocked. Overexpression of Rom2p results in delocalization of Rom2p and accumulation of 1,3-beta-glucan in secretory vesicles. Based on these results, we propose that Rho1p is kept inactive in intracellular secretory organelles, resulting in repression of the activity of the cell wall-synthesizing enzyme within cells.

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Artificial synthesis of 1,3-β-glucan by expression of activated RHO1 in secretory vesicles. (A) sec1 cells expressing the active form of RHO1. sec1 cells transformed with YCpU-RHO1 (G19V) were incubated at 37°C for 2 h, fixed by the freeze-substituted fixation method, and 1,3-β-glucan was detected by the mouse mAb to 1,3-β-glucan. Bar, 200 nm. (B) Wild-type cells expressing the active form of RHO1. Bar, 200 nm. (C) 1,3-β-Glucan in high-speed pellets. Cells were incubated at 37°C for 2 h. The high-spin pellet was resuspended and 10 μg protein was blotted. 1,3-β-Glucan was detected by the mouse mAb against 1,3-β-glucan. (D) Incorporation of [14C]glucose into 1,3-β-glucan in sec1 cells. Cells were cultured in the presence of [14C]glucose at 25°C (black bars) or at 34°C for 2 h (white bars).
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fig6: Artificial synthesis of 1,3-β-glucan by expression of activated RHO1 in secretory vesicles. (A) sec1 cells expressing the active form of RHO1. sec1 cells transformed with YCpU-RHO1 (G19V) were incubated at 37°C for 2 h, fixed by the freeze-substituted fixation method, and 1,3-β-glucan was detected by the mouse mAb to 1,3-β-glucan. Bar, 200 nm. (B) Wild-type cells expressing the active form of RHO1. Bar, 200 nm. (C) 1,3-β-Glucan in high-speed pellets. Cells were incubated at 37°C for 2 h. The high-spin pellet was resuspended and 10 μg protein was blotted. 1,3-β-Glucan was detected by the mouse mAb against 1,3-β-glucan. (D) Incorporation of [14C]glucose into 1,3-β-glucan in sec1 cells. Cells were cultured in the presence of [14C]glucose at 25°C (black bars) or at 34°C for 2 h (white bars).

Mentions: If accumulation of the inactive form of Rho1p results in impairment of 1,3-β-glucan synthesis in secretory vesicles, expression of active RHO1 must lead to synthesis of 1,3-β-glucan in secretory vesicles. Immunoelectron microscopic observations with the anti-1,3-β-glucan antibody revealed that 1,3-β-glucan accumulated in intracellular compartments after expression of the activated form of Rho1p (Fig. 6Figure 6.


Lack of GTP-bound Rho1p in secretory vesicles of Saccharomyces cerevisiae.

Abe M, Qadota H, Hirata A, Ohya Y - J. Cell Biol. (2003)

Artificial synthesis of 1,3-β-glucan by expression of activated RHO1 in secretory vesicles. (A) sec1 cells expressing the active form of RHO1. sec1 cells transformed with YCpU-RHO1 (G19V) were incubated at 37°C for 2 h, fixed by the freeze-substituted fixation method, and 1,3-β-glucan was detected by the mouse mAb to 1,3-β-glucan. Bar, 200 nm. (B) Wild-type cells expressing the active form of RHO1. Bar, 200 nm. (C) 1,3-β-Glucan in high-speed pellets. Cells were incubated at 37°C for 2 h. The high-spin pellet was resuspended and 10 μg protein was blotted. 1,3-β-Glucan was detected by the mouse mAb against 1,3-β-glucan. (D) Incorporation of [14C]glucose into 1,3-β-glucan in sec1 cells. Cells were cultured in the presence of [14C]glucose at 25°C (black bars) or at 34°C for 2 h (white bars).
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Related In: Results  -  Collection

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fig6: Artificial synthesis of 1,3-β-glucan by expression of activated RHO1 in secretory vesicles. (A) sec1 cells expressing the active form of RHO1. sec1 cells transformed with YCpU-RHO1 (G19V) were incubated at 37°C for 2 h, fixed by the freeze-substituted fixation method, and 1,3-β-glucan was detected by the mouse mAb to 1,3-β-glucan. Bar, 200 nm. (B) Wild-type cells expressing the active form of RHO1. Bar, 200 nm. (C) 1,3-β-Glucan in high-speed pellets. Cells were incubated at 37°C for 2 h. The high-spin pellet was resuspended and 10 μg protein was blotted. 1,3-β-Glucan was detected by the mouse mAb against 1,3-β-glucan. (D) Incorporation of [14C]glucose into 1,3-β-glucan in sec1 cells. Cells were cultured in the presence of [14C]glucose at 25°C (black bars) or at 34°C for 2 h (white bars).
Mentions: If accumulation of the inactive form of Rho1p results in impairment of 1,3-β-glucan synthesis in secretory vesicles, expression of active RHO1 must lead to synthesis of 1,3-β-glucan in secretory vesicles. Immunoelectron microscopic observations with the anti-1,3-β-glucan antibody revealed that 1,3-β-glucan accumulated in intracellular compartments after expression of the activated form of Rho1p (Fig. 6Figure 6.

Bottom Line: Rho1p, an essential Rho-type GTPase in Saccharomyces cerevisiae, activates its effectors in the GTP-bound form.Rom2p, the GDP/GTP exchange factor of Rho1p, is preferentially localized on the plasma membrane even when vesicular transport is blocked.Based on these results, we propose that Rho1p is kept inactive in intracellular secretory organelles, resulting in repression of the activity of the cell wall-synthesizing enzyme within cells.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, FSB-101, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan.

ABSTRACT
Rho1p, an essential Rho-type GTPase in Saccharomyces cerevisiae, activates its effectors in the GTP-bound form. Here, we show that Rho1p in secretory vesicles cannot activate 1,3-beta-glucan synthase, a cell wall synthesizing enzyme, during vesicular transport to the plasma membrane. Analyses with an antibody preferentially reacting with the GTP-bound form of Rho1p revealed that Rho1p remains in the inactive form in secretory vesicles. Rom2p, the GDP/GTP exchange factor of Rho1p, is preferentially localized on the plasma membrane even when vesicular transport is blocked. Overexpression of Rom2p results in delocalization of Rom2p and accumulation of 1,3-beta-glucan in secretory vesicles. Based on these results, we propose that Rho1p is kept inactive in intracellular secretory organelles, resulting in repression of the activity of the cell wall-synthesizing enzyme within cells.

Show MeSH
Related in: MedlinePlus