Limits...
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.

Show MeSH

Related in: MedlinePlus

Subcellular fractionation analysis of Rom2p when vesicular transport is blocked. (A) Distribution of Rom2p on the plasma membrane in wild-type cells. Wild-type cells were incubated in YPD at 37°C for 2 h. Cell lysate was fractionated on a 20–60% sucrose density gradient. Top, distribution of plasma membrane ATPase (open circles) and sucrose density (closed circles). Bottom, immunoblotting analysis of Rom2-HA with an anti-HA antibody. (B) Distribution of Rom2p in Sephacryl™ S-1000 column fractions of sec1 cells. Top, distribution of plasma membrane ATPase (closed circles) and invertase (open circles). Bottom, immunoblotting analysis of Rom2-HA.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172714&req=5

fig8: Subcellular fractionation analysis of Rom2p when vesicular transport is blocked. (A) Distribution of Rom2p on the plasma membrane in wild-type cells. Wild-type cells were incubated in YPD at 37°C for 2 h. Cell lysate was fractionated on a 20–60% sucrose density gradient. Top, distribution of plasma membrane ATPase (open circles) and sucrose density (closed circles). Bottom, immunoblotting analysis of Rom2-HA with an anti-HA antibody. (B) Distribution of Rom2p in Sephacryl™ S-1000 column fractions of sec1 cells. Top, distribution of plasma membrane ATPase (closed circles) and invertase (open circles). Bottom, immunoblotting analysis of Rom2-HA.

Mentions: To examine whether Rom2p is localized on the plasma membrane or secretory vesicles close to the plasma membrane, we isolated the plasma membrane from wild-type cells expressing ROM2-HA, and analyzed the distribution of the fusion protein. Immunoblotting analysis revealed that Rom2-HA is enriched in plasma membrane fractions (Fig. 8Figure 8.


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

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

Subcellular fractionation analysis of Rom2p when vesicular transport is blocked. (A) Distribution of Rom2p on the plasma membrane in wild-type cells. Wild-type cells were incubated in YPD at 37°C for 2 h. Cell lysate was fractionated on a 20–60% sucrose density gradient. Top, distribution of plasma membrane ATPase (open circles) and sucrose density (closed circles). Bottom, immunoblotting analysis of Rom2-HA with an anti-HA antibody. (B) Distribution of Rom2p in Sephacryl™ S-1000 column fractions of sec1 cells. Top, distribution of plasma membrane ATPase (closed circles) and invertase (open circles). Bottom, immunoblotting analysis of Rom2-HA.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Subcellular fractionation analysis of Rom2p when vesicular transport is blocked. (A) Distribution of Rom2p on the plasma membrane in wild-type cells. Wild-type cells were incubated in YPD at 37°C for 2 h. Cell lysate was fractionated on a 20–60% sucrose density gradient. Top, distribution of plasma membrane ATPase (open circles) and sucrose density (closed circles). Bottom, immunoblotting analysis of Rom2-HA with an anti-HA antibody. (B) Distribution of Rom2p in Sephacryl™ S-1000 column fractions of sec1 cells. Top, distribution of plasma membrane ATPase (closed circles) and invertase (open circles). Bottom, immunoblotting analysis of Rom2-HA.
Mentions: To examine whether Rom2p is localized on the plasma membrane or secretory vesicles close to the plasma membrane, we isolated the plasma membrane from wild-type cells expressing ROM2-HA, and analyzed the distribution of the fusion protein. Immunoblotting analysis revealed that Rom2-HA is enriched in plasma membrane fractions (Fig. 8Figure 8.

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