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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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Secretory vesicle fractions ofsec1mutant and wild-type cells. Wild-type (left) or sec1 (right) cells were incubated at 37°C for 2 h, lysed, and subjected to differential centrifugations. The high-spin pellet was applied to a Sephacryl™ S-1000 column, and 4-ml fractions were collected. (A) Distributions of plasma membrane ATPase (closed circles) and invertase activity (open circles). (B) Immunoblotting analysis of GS-containing fractions. The amounts of Rho1p and Fks1p/2p were estimated with the guinea pig antiserum against Rho1p and the mouse mAb against Fks1p/2p, respectively. (C) Distributions of GS activity in the presence of GTP-γS.
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fig2: Secretory vesicle fractions ofsec1mutant and wild-type cells. Wild-type (left) or sec1 (right) cells were incubated at 37°C for 2 h, lysed, and subjected to differential centrifugations. The high-spin pellet was applied to a Sephacryl™ S-1000 column, and 4-ml fractions were collected. (A) Distributions of plasma membrane ATPase (closed circles) and invertase activity (open circles). (B) Immunoblotting analysis of GS-containing fractions. The amounts of Rho1p and Fks1p/2p were estimated with the guinea pig antiserum against Rho1p and the mouse mAb against Fks1p/2p, respectively. (C) Distributions of GS activity in the presence of GTP-γS.

Mentions: We confirmed by cell fractionations that movement of nascent GS is attributable to vesicular transport. Secretory vesicles were isolated from sec1 cells cultured at the restrictive temperature for 2 h after growth at the permissive temperature and were used to examine whether Rho1p and Fks1p/2p are detected in secretory vesicle fractions. As described previously (Walworth and Novick, 1987; McCaffrey et al., 1991), sec1 cell lysate was subjected to differential centrifugations, and the high-speed pellet obtained was fractionated further on the basis of vesicular size by gel exclusion chromatography. First, we examined the distribution of marker enzymes in the final fractions. Invertase, a marker enzyme of secretory vesicles, was eluted from the column as a single peak with its maximum at fraction 23 (Fig. 2Figure 2.


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

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

Secretory vesicle fractions ofsec1mutant and wild-type cells. Wild-type (left) or sec1 (right) cells were incubated at 37°C for 2 h, lysed, and subjected to differential centrifugations. The high-spin pellet was applied to a Sephacryl™ S-1000 column, and 4-ml fractions were collected. (A) Distributions of plasma membrane ATPase (closed circles) and invertase activity (open circles). (B) Immunoblotting analysis of GS-containing fractions. The amounts of Rho1p and Fks1p/2p were estimated with the guinea pig antiserum against Rho1p and the mouse mAb against Fks1p/2p, respectively. (C) Distributions of GS activity in the presence of GTP-γS.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Secretory vesicle fractions ofsec1mutant and wild-type cells. Wild-type (left) or sec1 (right) cells were incubated at 37°C for 2 h, lysed, and subjected to differential centrifugations. The high-spin pellet was applied to a Sephacryl™ S-1000 column, and 4-ml fractions were collected. (A) Distributions of plasma membrane ATPase (closed circles) and invertase activity (open circles). (B) Immunoblotting analysis of GS-containing fractions. The amounts of Rho1p and Fks1p/2p were estimated with the guinea pig antiserum against Rho1p and the mouse mAb against Fks1p/2p, respectively. (C) Distributions of GS activity in the presence of GTP-γS.
Mentions: We confirmed by cell fractionations that movement of nascent GS is attributable to vesicular transport. Secretory vesicles were isolated from sec1 cells cultured at the restrictive temperature for 2 h after growth at the permissive temperature and were used to examine whether Rho1p and Fks1p/2p are detected in secretory vesicle fractions. As described previously (Walworth and Novick, 1987; McCaffrey et al., 1991), sec1 cell lysate was subjected to differential centrifugations, and the high-speed pellet obtained was fractionated further on the basis of vesicular size by gel exclusion chromatography. First, we examined the distribution of marker enzymes in the final fractions. Invertase, a marker enzyme of secretory vesicles, was eluted from the column as a single peak with its maximum at fraction 23 (Fig. 2Figure 2.

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