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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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1,3-β-Glucan is not synthesized in intracellular organelles. (A) Immunogold labeling with the anti-1,3-β-glucan antibody in sec1 cells shifted to 37°C and cultured for 2 h. Bar, 1 μm. (B) The magnification image. Bar, 200 nm. (C) Reduced incorporation of glucose into 1,3-β-glucan in sec cells. Cells were cultured in YPD in the presence of [14C]glucose either at 25°C (black bars) or at 34°C for 2 h (white bars). The data represent the means and SDs of at least three experiments. (D) GS activity of the membrane fractions isolated from sec mutant cells. Cells were cultured either at 25°C (black bars) or at 37°C for 2 h (white bars), from which membrane fractions were isolated and assayed for GS activity in the presence of UDP-[14C]glucose and GTP-γS. The data represent the means and SDs of at least four experiments.
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fig3: 1,3-β-Glucan is not synthesized in intracellular organelles. (A) Immunogold labeling with the anti-1,3-β-glucan antibody in sec1 cells shifted to 37°C and cultured for 2 h. Bar, 1 μm. (B) The magnification image. Bar, 200 nm. (C) Reduced incorporation of glucose into 1,3-β-glucan in sec cells. Cells were cultured in YPD in the presence of [14C]glucose either at 25°C (black bars) or at 34°C for 2 h (white bars). The data represent the means and SDs of at least three experiments. (D) GS activity of the membrane fractions isolated from sec mutant cells. Cells were cultured either at 25°C (black bars) or at 37°C for 2 h (white bars), from which membrane fractions were isolated and assayed for GS activity in the presence of UDP-[14C]glucose and GTP-γS. The data represent the means and SDs of at least four experiments.

Mentions: To identify where in vesicular transport steps nascent GS becomes activated, we examined whether 1,3-β-glucan is detected in intracellular organelles. An immunoelectron microscopic observation with an anti-1,3-β-glucan antibody revealed that 1,3-β-glucan is not detected in any organelles other than cell wall layers in wild-type cells (unpublished data). In addition, after shift to the restrictive temperature, 1,3-β-glucan was not detected in secretory vesicles accumulated in sec1 cells, but it was observed in cell wall layers (Fig. 3, A and BFigure 3.


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

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

1,3-β-Glucan is not synthesized in intracellular organelles. (A) Immunogold labeling with the anti-1,3-β-glucan antibody in sec1 cells shifted to 37°C and cultured for 2 h. Bar, 1 μm. (B) The magnification image. Bar, 200 nm. (C) Reduced incorporation of glucose into 1,3-β-glucan in sec cells. Cells were cultured in YPD in the presence of [14C]glucose either at 25°C (black bars) or at 34°C for 2 h (white bars). The data represent the means and SDs of at least three experiments. (D) GS activity of the membrane fractions isolated from sec mutant cells. Cells were cultured either at 25°C (black bars) or at 37°C for 2 h (white bars), from which membrane fractions were isolated and assayed for GS activity in the presence of UDP-[14C]glucose and GTP-γS. The data represent the means and SDs of at least four experiments.
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Related In: Results  -  Collection

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fig3: 1,3-β-Glucan is not synthesized in intracellular organelles. (A) Immunogold labeling with the anti-1,3-β-glucan antibody in sec1 cells shifted to 37°C and cultured for 2 h. Bar, 1 μm. (B) The magnification image. Bar, 200 nm. (C) Reduced incorporation of glucose into 1,3-β-glucan in sec cells. Cells were cultured in YPD in the presence of [14C]glucose either at 25°C (black bars) or at 34°C for 2 h (white bars). The data represent the means and SDs of at least three experiments. (D) GS activity of the membrane fractions isolated from sec mutant cells. Cells were cultured either at 25°C (black bars) or at 37°C for 2 h (white bars), from which membrane fractions were isolated and assayed for GS activity in the presence of UDP-[14C]glucose and GTP-γS. The data represent the means and SDs of at least four experiments.
Mentions: To identify where in vesicular transport steps nascent GS becomes activated, we examined whether 1,3-β-glucan is detected in intracellular organelles. An immunoelectron microscopic observation with an anti-1,3-β-glucan antibody revealed that 1,3-β-glucan is not detected in any organelles other than cell wall layers in wild-type cells (unpublished data). In addition, after shift to the restrictive temperature, 1,3-β-glucan was not detected in secretory vesicles accumulated in sec1 cells, but it was observed in cell wall layers (Fig. 3, A and BFigure 3.

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