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Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud.

Adamo JE, Moskow JJ, Gladfelter AS, Viterbo D, Lew DJ, Brennwald PJ - J. Cell Biol. (2001)

Bottom Line: The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells.Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis.Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

ABSTRACT
The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. In yeast, the role of Cdc42 in polarization of cell growth includes polarization of the actin cytoskeleton, which delivers secretory vesicles to growth sites at the plasma membrane. We now describe a novel temperature-sensitive mutant, cdc42-6, that reveals a role for Cdc42 in docking and fusion of secretory vesicles that is independent of its role in actin polarization. cdc42-6 mutants can polarize actin and deliver secretory vesicles to the bud, but fail to fuse those vesicles with the plasma membrane. This defect is manifested only during the early stages of bud formation when growth is most highly polarized, and appears to reflect a requirement for Cdc42 to maintain maximally active exocytic machinery at sites of high vesicle throughput. Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis. Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus. Rather, we suggest that Cdc42 acts as an allosteric regulator of the vesicle docking and fusion apparatus to provide maximal function at sites of polarized growth.

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Trafficking of Bgl2, invertase, and CPY in the cdc42-6 mutant. (A) The graph depicts the percentage of Bgl2 protein that remains internal in CDC42, cdc42-6, and sec6-4 strains. The percent distribution of Bgl2 was determined by immunoblot analysis using affinity-purified antibodies raised against the COOH terminus of Bgl2. Quantitation of the bands was done using ImageQuant software and the results are presented as a percentage of total Bgl2 that is found internally. (B) The graph depicts the percentage of invertase protein that remains internal in CDC42, cdc42-6, and sec6-4 strains. The percent of internal invertase was determined by enzyme assays and then shown in bar graph form. (C) Transport of the vacuolar protein CPY is not affected in cdc42-6, as it is found exclusively in the mature form, (mCPY, 61 kD). Wild-type CDC42 and the late secretory mutant, sec6-4, are also shown to process CPY fully to the mature form while sec18-1, an ER-to-Golgi mutant, accumulates the earlier forms. (D) Vesicle accumulation was quantitated for wild-type and cdc42-6 cells at both permissive and restrictive (33°C for 1 h) temperatures from randomly chosen fields of cells. Numbers shown were determined by dividing the total number of 80–100-nm vesicles observed per field of cells by the total number of cells present per field.
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fig3: Trafficking of Bgl2, invertase, and CPY in the cdc42-6 mutant. (A) The graph depicts the percentage of Bgl2 protein that remains internal in CDC42, cdc42-6, and sec6-4 strains. The percent distribution of Bgl2 was determined by immunoblot analysis using affinity-purified antibodies raised against the COOH terminus of Bgl2. Quantitation of the bands was done using ImageQuant software and the results are presented as a percentage of total Bgl2 that is found internally. (B) The graph depicts the percentage of invertase protein that remains internal in CDC42, cdc42-6, and sec6-4 strains. The percent of internal invertase was determined by enzyme assays and then shown in bar graph form. (C) Transport of the vacuolar protein CPY is not affected in cdc42-6, as it is found exclusively in the mature form, (mCPY, 61 kD). Wild-type CDC42 and the late secretory mutant, sec6-4, are also shown to process CPY fully to the mature form while sec18-1, an ER-to-Golgi mutant, accumulates the earlier forms. (D) Vesicle accumulation was quantitated for wild-type and cdc42-6 cells at both permissive and restrictive (33°C for 1 h) temperatures from randomly chosen fields of cells. Numbers shown were determined by dividing the total number of 80–100-nm vesicles observed per field of cells by the total number of cells present per field.

Mentions: To directly determine whether cdc42-6 mutants had a secretory defect, we initially focused on two proteins secreted into the periplasmic space, the abundant exoglucanase Bgl2 and the sucrose cleaving enzyme invertase. These two enzymes were chosen because they represent well-characterized markers for each of the two major classes of post-Golgi vesicles that deliver proteins to the cell surface (Harsay and Bretscher, 1995). Surprisingly, secretion of invertase was not detectably perturbed in cdc42-6 cells even at restrictive temperature, in contrast to the control sec6-4 cells (Fig. 3 A). However, a very clear effect was apparent when we examined the secretion of Bgl2 (Fig. 3 B). After a 1-h shift to 33°C, there was a pronounced effect on the accumulation of internal Bgl2 in cdc42-6 cells, to a level even higher than that seen in the control sec6-4 cells. Unlike sec6-4 cells, cdc42-6 cells also have a detectable Bgl2 secretory defect at permissive temperature; however, the magnitude of the defect increased significantly following the 1-h shift to 33°C (Fig. 3 B). Consistent with the secretory defects observed for Bgl2, analysis of cdc42-6 cells by electron microscopy revealed that these cells accumulated 80–100-nm vesicles in a temperature-sensitive manner, with some accumulation of vesicles at permissive conditions but a large increase following a 1-h shift to 33°C (Fig. 3 D). In addition to an overall increase in numbers of vesicles per cell, the penetrance of the vesicle accumulation phenotype also increased following the temperature shift. This is discussed in greater detail below.


Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud.

Adamo JE, Moskow JJ, Gladfelter AS, Viterbo D, Lew DJ, Brennwald PJ - J. Cell Biol. (2001)

Trafficking of Bgl2, invertase, and CPY in the cdc42-6 mutant. (A) The graph depicts the percentage of Bgl2 protein that remains internal in CDC42, cdc42-6, and sec6-4 strains. The percent distribution of Bgl2 was determined by immunoblot analysis using affinity-purified antibodies raised against the COOH terminus of Bgl2. Quantitation of the bands was done using ImageQuant software and the results are presented as a percentage of total Bgl2 that is found internally. (B) The graph depicts the percentage of invertase protein that remains internal in CDC42, cdc42-6, and sec6-4 strains. The percent of internal invertase was determined by enzyme assays and then shown in bar graph form. (C) Transport of the vacuolar protein CPY is not affected in cdc42-6, as it is found exclusively in the mature form, (mCPY, 61 kD). Wild-type CDC42 and the late secretory mutant, sec6-4, are also shown to process CPY fully to the mature form while sec18-1, an ER-to-Golgi mutant, accumulates the earlier forms. (D) Vesicle accumulation was quantitated for wild-type and cdc42-6 cells at both permissive and restrictive (33°C for 1 h) temperatures from randomly chosen fields of cells. Numbers shown were determined by dividing the total number of 80–100-nm vesicles observed per field of cells by the total number of cells present per field.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198861&req=5

fig3: Trafficking of Bgl2, invertase, and CPY in the cdc42-6 mutant. (A) The graph depicts the percentage of Bgl2 protein that remains internal in CDC42, cdc42-6, and sec6-4 strains. The percent distribution of Bgl2 was determined by immunoblot analysis using affinity-purified antibodies raised against the COOH terminus of Bgl2. Quantitation of the bands was done using ImageQuant software and the results are presented as a percentage of total Bgl2 that is found internally. (B) The graph depicts the percentage of invertase protein that remains internal in CDC42, cdc42-6, and sec6-4 strains. The percent of internal invertase was determined by enzyme assays and then shown in bar graph form. (C) Transport of the vacuolar protein CPY is not affected in cdc42-6, as it is found exclusively in the mature form, (mCPY, 61 kD). Wild-type CDC42 and the late secretory mutant, sec6-4, are also shown to process CPY fully to the mature form while sec18-1, an ER-to-Golgi mutant, accumulates the earlier forms. (D) Vesicle accumulation was quantitated for wild-type and cdc42-6 cells at both permissive and restrictive (33°C for 1 h) temperatures from randomly chosen fields of cells. Numbers shown were determined by dividing the total number of 80–100-nm vesicles observed per field of cells by the total number of cells present per field.
Mentions: To directly determine whether cdc42-6 mutants had a secretory defect, we initially focused on two proteins secreted into the periplasmic space, the abundant exoglucanase Bgl2 and the sucrose cleaving enzyme invertase. These two enzymes were chosen because they represent well-characterized markers for each of the two major classes of post-Golgi vesicles that deliver proteins to the cell surface (Harsay and Bretscher, 1995). Surprisingly, secretion of invertase was not detectably perturbed in cdc42-6 cells even at restrictive temperature, in contrast to the control sec6-4 cells (Fig. 3 A). However, a very clear effect was apparent when we examined the secretion of Bgl2 (Fig. 3 B). After a 1-h shift to 33°C, there was a pronounced effect on the accumulation of internal Bgl2 in cdc42-6 cells, to a level even higher than that seen in the control sec6-4 cells. Unlike sec6-4 cells, cdc42-6 cells also have a detectable Bgl2 secretory defect at permissive temperature; however, the magnitude of the defect increased significantly following the 1-h shift to 33°C (Fig. 3 B). Consistent with the secretory defects observed for Bgl2, analysis of cdc42-6 cells by electron microscopy revealed that these cells accumulated 80–100-nm vesicles in a temperature-sensitive manner, with some accumulation of vesicles at permissive conditions but a large increase following a 1-h shift to 33°C (Fig. 3 D). In addition to an overall increase in numbers of vesicles per cell, the penetrance of the vesicle accumulation phenotype also increased following the temperature shift. This is discussed in greater detail below.

Bottom Line: The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells.Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis.Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

ABSTRACT
The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. In yeast, the role of Cdc42 in polarization of cell growth includes polarization of the actin cytoskeleton, which delivers secretory vesicles to growth sites at the plasma membrane. We now describe a novel temperature-sensitive mutant, cdc42-6, that reveals a role for Cdc42 in docking and fusion of secretory vesicles that is independent of its role in actin polarization. cdc42-6 mutants can polarize actin and deliver secretory vesicles to the bud, but fail to fuse those vesicles with the plasma membrane. This defect is manifested only during the early stages of bud formation when growth is most highly polarized, and appears to reflect a requirement for Cdc42 to maintain maximally active exocytic machinery at sites of high vesicle throughput. Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis. Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus. Rather, we suggest that Cdc42 acts as an allosteric regulator of the vesicle docking and fusion apparatus to provide maximal function at sites of polarized growth.

Show MeSH
Related in: MedlinePlus