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

Rom2p is localized on the plasma membrane even when vesicular transport is blocked. (A–D) Localization of Rom2-GFP fusion protein. rom2Δ cells transformed with YCpU-ROM2-GFP were incubated at 37°C for 2 h (A). sec1 rom2Δ cells transformed with YCpU-ROM2-GFP were incubated at 37°C for 2 h in the absence (B) or presence (C) of cycloheximide. sec1 rom2Δ cells transformed with YEpU-ROM2-GFP were incubated at 37°C for 2 h (D). Cultured cells were all fixed in methanol, and were subjected to observation. (E) Immunogold labeling with the anti-1,3-β-glucan antibody in sec1 cells overexpressing ROM2. sec1 cells were transformed with YEpU-ROM2. Transformants were incubated at 37°C for 2 h, and 1,3-β-glucan was detected. Bar, 200 nm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172714&req=5

fig7: Rom2p is localized on the plasma membrane even when vesicular transport is blocked. (A–D) Localization of Rom2-GFP fusion protein. rom2Δ cells transformed with YCpU-ROM2-GFP were incubated at 37°C for 2 h (A). sec1 rom2Δ cells transformed with YCpU-ROM2-GFP were incubated at 37°C for 2 h in the absence (B) or presence (C) of cycloheximide. sec1 rom2Δ cells transformed with YEpU-ROM2-GFP were incubated at 37°C for 2 h (D). Cultured cells were all fixed in methanol, and were subjected to observation. (E) Immunogold labeling with the anti-1,3-β-glucan antibody in sec1 cells overexpressing ROM2. sec1 cells were transformed with YEpU-ROM2. Transformants were incubated at 37°C for 2 h, and 1,3-β-glucan was detected. Bar, 200 nm.

Mentions: To test the possibility that Wsc1p or Rom2p is involved in the inactivation of Rho1p in secretory vesicles, we checked the localization of Wsc1p and Rom2p in sec1 cells at the restrictive temperature. We constructed Wsc1p and Rom2p with an HA or a GFP tag at the COOH terminus. All the fusion proteins were judged to be functional because the temperature-sensitive growth defect as well as the cell lysis phenotype in wsc1Δ and rom2Δ cells was suppressed by expression of Wsc1p and Rom2p fusion proteins, respectively (unpublished data). Next, we examined the subcellular localization of Wsc1-GFP and Rom2-GFP by direct microscopic observations. As described previously (Verna et al., 1997), Wsc1-GFP was localized at the site of growth in wild-type and sec1 cells at 25°C, whereas Wsc1-GFP was distributed all around cells of the sec1 mutant at the restrictive temperature, (unpublished data). These results suggested that Wsc1-GFP accumulates in secretory vesicles when vesicular transport is blocked. As formerly reported (Manning et al., 1997; Audhya and Emr, 2002), Rom2-GFP was localized at the site of growth in wild-type and sec1 cells at 25°C (unpublished data). Interestingly, culturing at 37°C for 2 h did not alter the localization of Rom2-GFP both in wild-type (Fig. 7Figure 7.


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

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

Rom2p is localized on the plasma membrane even when vesicular transport is blocked. (A–D) Localization of Rom2-GFP fusion protein. rom2Δ cells transformed with YCpU-ROM2-GFP were incubated at 37°C for 2 h (A). sec1 rom2Δ cells transformed with YCpU-ROM2-GFP were incubated at 37°C for 2 h in the absence (B) or presence (C) of cycloheximide. sec1 rom2Δ cells transformed with YEpU-ROM2-GFP were incubated at 37°C for 2 h (D). Cultured cells were all fixed in methanol, and were subjected to observation. (E) Immunogold labeling with the anti-1,3-β-glucan antibody in sec1 cells overexpressing ROM2. sec1 cells were transformed with YEpU-ROM2. Transformants were incubated at 37°C for 2 h, and 1,3-β-glucan was detected. Bar, 200 nm.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Rom2p is localized on the plasma membrane even when vesicular transport is blocked. (A–D) Localization of Rom2-GFP fusion protein. rom2Δ cells transformed with YCpU-ROM2-GFP were incubated at 37°C for 2 h (A). sec1 rom2Δ cells transformed with YCpU-ROM2-GFP were incubated at 37°C for 2 h in the absence (B) or presence (C) of cycloheximide. sec1 rom2Δ cells transformed with YEpU-ROM2-GFP were incubated at 37°C for 2 h (D). Cultured cells were all fixed in methanol, and were subjected to observation. (E) Immunogold labeling with the anti-1,3-β-glucan antibody in sec1 cells overexpressing ROM2. sec1 cells were transformed with YEpU-ROM2. Transformants were incubated at 37°C for 2 h, and 1,3-β-glucan was detected. Bar, 200 nm.
Mentions: To test the possibility that Wsc1p or Rom2p is involved in the inactivation of Rho1p in secretory vesicles, we checked the localization of Wsc1p and Rom2p in sec1 cells at the restrictive temperature. We constructed Wsc1p and Rom2p with an HA or a GFP tag at the COOH terminus. All the fusion proteins were judged to be functional because the temperature-sensitive growth defect as well as the cell lysis phenotype in wsc1Δ and rom2Δ cells was suppressed by expression of Wsc1p and Rom2p fusion proteins, respectively (unpublished data). Next, we examined the subcellular localization of Wsc1-GFP and Rom2-GFP by direct microscopic observations. As described previously (Verna et al., 1997), Wsc1-GFP was localized at the site of growth in wild-type and sec1 cells at 25°C, whereas Wsc1-GFP was distributed all around cells of the sec1 mutant at the restrictive temperature, (unpublished data). These results suggested that Wsc1-GFP accumulates in secretory vesicles when vesicular transport is blocked. As formerly reported (Manning et al., 1997; Audhya and Emr, 2002), Rom2-GFP was localized at the site of growth in wild-type and sec1 cells at 25°C (unpublished data). Interestingly, culturing at 37°C for 2 h did not alter the localization of Rom2-GFP both in wild-type (Fig. 7Figure 7.

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