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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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Distribution of wild-type Rho1p and activated Rho1p. (A) Colocalization of wild-type Rho1p and Fks1p/2p. Wild-type cells were cultured at 25°C, fixed, and stained for immunofluorescence microscopy with the anti-Fks1p/2p antibody (green) or the anti-Rho1p antibody (red). (B) Localization of activated Rho1p to a restricted region on the plasma membrane in wild-type cells. Wild-type cells were cultured at 25°C (top panels), whereas sec1 mutant cells were incubated at 37°C for 2 h (bottom panels), and stained for immunofluorescence microscopy with the anti-Fks1p/2p antibody (green) or the anti-actRho1p antibody (red).
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fig5: Distribution of wild-type Rho1p and activated Rho1p. (A) Colocalization of wild-type Rho1p and Fks1p/2p. Wild-type cells were cultured at 25°C, fixed, and stained for immunofluorescence microscopy with the anti-Fks1p/2p antibody (green) or the anti-Rho1p antibody (red). (B) Localization of activated Rho1p to a restricted region on the plasma membrane in wild-type cells. Wild-type cells were cultured at 25°C (top panels), whereas sec1 mutant cells were incubated at 37°C for 2 h (bottom panels), and stained for immunofluorescence microscopy with the anti-Fks1p/2p antibody (green) or the anti-actRho1p antibody (red).

Mentions: To verify this, we examined the localization of activated Rho1p with the antibody by immunofluorescence microscopy. We incubated wild-type and sec1 cells for 2 h at 25 and 37°C, respectively, and subsequently observed the localization of activated Rho1p. In wild-type cells, Rho1p was detected either in a large region in a small or large bud or at the site of bud emergence in an unbudded cell, and in either case, mostly colocalized with Fks1p/2p (Fig. 5Figure 5.


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

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

Distribution of wild-type Rho1p and activated Rho1p. (A) Colocalization of wild-type Rho1p and Fks1p/2p. Wild-type cells were cultured at 25°C, fixed, and stained for immunofluorescence microscopy with the anti-Fks1p/2p antibody (green) or the anti-Rho1p antibody (red). (B) Localization of activated Rho1p to a restricted region on the plasma membrane in wild-type cells. Wild-type cells were cultured at 25°C (top panels), whereas sec1 mutant cells were incubated at 37°C for 2 h (bottom panels), and stained for immunofluorescence microscopy with the anti-Fks1p/2p antibody (green) or the anti-actRho1p antibody (red).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172714&req=5

fig5: Distribution of wild-type Rho1p and activated Rho1p. (A) Colocalization of wild-type Rho1p and Fks1p/2p. Wild-type cells were cultured at 25°C, fixed, and stained for immunofluorescence microscopy with the anti-Fks1p/2p antibody (green) or the anti-Rho1p antibody (red). (B) Localization of activated Rho1p to a restricted region on the plasma membrane in wild-type cells. Wild-type cells were cultured at 25°C (top panels), whereas sec1 mutant cells were incubated at 37°C for 2 h (bottom panels), and stained for immunofluorescence microscopy with the anti-Fks1p/2p antibody (green) or the anti-actRho1p antibody (red).
Mentions: To verify this, we examined the localization of activated Rho1p with the antibody by immunofluorescence microscopy. We incubated wild-type and sec1 cells for 2 h at 25 and 37°C, respectively, and subsequently observed the localization of activated Rho1p. In wild-type cells, Rho1p was detected either in a large region in a small or large bud or at the site of bud emergence in an unbudded cell, and in either case, mostly colocalized with Fks1p/2p (Fig. 5Figure 5.

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