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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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Localization of activated Rho1p detected with the purified anti-actRho1p antibody. (A) Affinity purification of the anti-actRho1p antibody. Antibody was raised against purified recombinant Rho1 (G19V) protein. Antiserum was loaded on an affinity column to which Rho1 (G19V) protein had been bound. The bound antibody was eluted, applied to another column charged with wild-type Rho1 protein, and the flow-through fractions were collected. (B) Immunoprecipitation of Rho1p with the anti-Rho1p (left), or the anti-actRho1p (right) antibody in the presence (+) or absence (−) of GDP and GTP-γS. Rho1p was detected by immunoblotting analysis with the guinea pig antiserum against Rho1p. (C) Immunoprecipitation of activated Rho1p. Wild-type (left) or sec1 mutant (right) cells were incubated at 37°C for 2 h. The cells were lysed in the presence or absence of 4 μM GTP, and fractionated by differential centrifugations. The high-spin pellet was applied to a Sephacryl™ S-1000 column without GTP, and 3-ml fractions were collected. Top panels, distribution of plasma membrane ATPase (closed circles) and invertase (open circles). Bottom panels, the distributions of activated Rho1p (closed circles and triangles) and total Rho1p (open circles). Aliquots of each fraction were assayed by immunoprecipitation with the anti-actRho1p, and detected by immunoblotting analysis with the guinea pig antiserum against Rho1p. The relative amount was quantified with a cooled CCD camera (LAS-1000plus; Fuji Photo Film).
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fig4: Localization of activated Rho1p detected with the purified anti-actRho1p antibody. (A) Affinity purification of the anti-actRho1p antibody. Antibody was raised against purified recombinant Rho1 (G19V) protein. Antiserum was loaded on an affinity column to which Rho1 (G19V) protein had been bound. The bound antibody was eluted, applied to another column charged with wild-type Rho1 protein, and the flow-through fractions were collected. (B) Immunoprecipitation of Rho1p with the anti-Rho1p (left), or the anti-actRho1p (right) antibody in the presence (+) or absence (−) of GDP and GTP-γS. Rho1p was detected by immunoblotting analysis with the guinea pig antiserum against Rho1p. (C) Immunoprecipitation of activated Rho1p. Wild-type (left) or sec1 mutant (right) cells were incubated at 37°C for 2 h. The cells were lysed in the presence or absence of 4 μM GTP, and fractionated by differential centrifugations. The high-spin pellet was applied to a Sephacryl™ S-1000 column without GTP, and 3-ml fractions were collected. Top panels, distribution of plasma membrane ATPase (closed circles) and invertase (open circles). Bottom panels, the distributions of activated Rho1p (closed circles and triangles) and total Rho1p (open circles). Aliquots of each fraction were assayed by immunoprecipitation with the anti-actRho1p, and detected by immunoblotting analysis with the guinea pig antiserum against Rho1p. The relative amount was quantified with a cooled CCD camera (LAS-1000plus; Fuji Photo Film).

Mentions: To directly examine the dearth of the GTP-bound form of Rho1p in secretory vesicles, we produced an antibody that preferentially reacts with the GTP-bound form of Rho1p (Fig. 4Figure 4.


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

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

Localization of activated Rho1p detected with the purified anti-actRho1p antibody. (A) Affinity purification of the anti-actRho1p antibody. Antibody was raised against purified recombinant Rho1 (G19V) protein. Antiserum was loaded on an affinity column to which Rho1 (G19V) protein had been bound. The bound antibody was eluted, applied to another column charged with wild-type Rho1 protein, and the flow-through fractions were collected. (B) Immunoprecipitation of Rho1p with the anti-Rho1p (left), or the anti-actRho1p (right) antibody in the presence (+) or absence (−) of GDP and GTP-γS. Rho1p was detected by immunoblotting analysis with the guinea pig antiserum against Rho1p. (C) Immunoprecipitation of activated Rho1p. Wild-type (left) or sec1 mutant (right) cells were incubated at 37°C for 2 h. The cells were lysed in the presence or absence of 4 μM GTP, and fractionated by differential centrifugations. The high-spin pellet was applied to a Sephacryl™ S-1000 column without GTP, and 3-ml fractions were collected. Top panels, distribution of plasma membrane ATPase (closed circles) and invertase (open circles). Bottom panels, the distributions of activated Rho1p (closed circles and triangles) and total Rho1p (open circles). Aliquots of each fraction were assayed by immunoprecipitation with the anti-actRho1p, and detected by immunoblotting analysis with the guinea pig antiserum against Rho1p. The relative amount was quantified with a cooled CCD camera (LAS-1000plus; Fuji Photo Film).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172714&req=5

fig4: Localization of activated Rho1p detected with the purified anti-actRho1p antibody. (A) Affinity purification of the anti-actRho1p antibody. Antibody was raised against purified recombinant Rho1 (G19V) protein. Antiserum was loaded on an affinity column to which Rho1 (G19V) protein had been bound. The bound antibody was eluted, applied to another column charged with wild-type Rho1 protein, and the flow-through fractions were collected. (B) Immunoprecipitation of Rho1p with the anti-Rho1p (left), or the anti-actRho1p (right) antibody in the presence (+) or absence (−) of GDP and GTP-γS. Rho1p was detected by immunoblotting analysis with the guinea pig antiserum against Rho1p. (C) Immunoprecipitation of activated Rho1p. Wild-type (left) or sec1 mutant (right) cells were incubated at 37°C for 2 h. The cells were lysed in the presence or absence of 4 μM GTP, and fractionated by differential centrifugations. The high-spin pellet was applied to a Sephacryl™ S-1000 column without GTP, and 3-ml fractions were collected. Top panels, distribution of plasma membrane ATPase (closed circles) and invertase (open circles). Bottom panels, the distributions of activated Rho1p (closed circles and triangles) and total Rho1p (open circles). Aliquots of each fraction were assayed by immunoprecipitation with the anti-actRho1p, and detected by immunoblotting analysis with the guinea pig antiserum against Rho1p. The relative amount was quantified with a cooled CCD camera (LAS-1000plus; Fuji Photo Film).
Mentions: To directly examine the dearth of the GTP-bound form of Rho1p in secretory vesicles, we produced an antibody that preferentially reacts with the GTP-bound form of Rho1p (Fig. 4Figure 4.

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