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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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Levels of Cdc42 accumulation and localization of Cdc42 and Sec4 in CDC42, cdc42-6 and cdc42-1 strains. (A) Whole cell lysates were prepared and subjected to SDS-PAGE analysis and then blotted with anti-Cdc42 or anti-Sso2 antibodies. (B) Immunofluorescence probing for Sec4 and Cdc42 localization was done after a shift to the restrictive temperature of 33°C for CDC42 (top), cdc42-6 (middle) and to 37°C for cdc42-1 (bottom). Bar, 5 μm.
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fig8: Levels of Cdc42 accumulation and localization of Cdc42 and Sec4 in CDC42, cdc42-6 and cdc42-1 strains. (A) Whole cell lysates were prepared and subjected to SDS-PAGE analysis and then blotted with anti-Cdc42 or anti-Sso2 antibodies. (B) Immunofluorescence probing for Sec4 and Cdc42 localization was done after a shift to the restrictive temperature of 33°C for CDC42 (top), cdc42-6 (middle) and to 37°C for cdc42-1 (bottom). Bar, 5 μm.

Mentions: Immunoblot analysis indicated that there was a slight decrease in the amount of Cdc42-6 protein at 33°C and a slightly greater decrease at 37°C, but the change in proteins levels alone did not appear to account for the thermosensitivity of this allele (Fig. 8 A). This is especially clear when compared with the levels of protein present in a cdc42-1 mutant, which shows drastically reduced amounts of the protein at all temperatures, consistent with previous analyses, but which grows normally at permissive temperature. Indirect immunofluorescence analysis of CDC42, cdc42-1, and cdc42-6 strains after a 1-h shift to restrictive temperature (33°C) indicated that unlike Cdc42-1 which was only detectable in 15% of small-budded cells, Cdc42-6 was clearly polarized to the bud tip in 78% of small-budded cells, virtually the same as wild-type Cdc42 (Fig. 8 B). We conclude that the phenotype of the cdc42-6 cells is unlikely to be a result of mislocalization of the mutant Cdc42-6 protein, but rather an inability of the mutant protein to carry out some other function.


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)

Levels of Cdc42 accumulation and localization of Cdc42 and Sec4 in CDC42, cdc42-6 and cdc42-1 strains. (A) Whole cell lysates were prepared and subjected to SDS-PAGE analysis and then blotted with anti-Cdc42 or anti-Sso2 antibodies. (B) Immunofluorescence probing for Sec4 and Cdc42 localization was done after a shift to the restrictive temperature of 33°C for CDC42 (top), cdc42-6 (middle) and to 37°C for cdc42-1 (bottom). Bar, 5 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198861&req=5

fig8: Levels of Cdc42 accumulation and localization of Cdc42 and Sec4 in CDC42, cdc42-6 and cdc42-1 strains. (A) Whole cell lysates were prepared and subjected to SDS-PAGE analysis and then blotted with anti-Cdc42 or anti-Sso2 antibodies. (B) Immunofluorescence probing for Sec4 and Cdc42 localization was done after a shift to the restrictive temperature of 33°C for CDC42 (top), cdc42-6 (middle) and to 37°C for cdc42-1 (bottom). Bar, 5 μm.
Mentions: Immunoblot analysis indicated that there was a slight decrease in the amount of Cdc42-6 protein at 33°C and a slightly greater decrease at 37°C, but the change in proteins levels alone did not appear to account for the thermosensitivity of this allele (Fig. 8 A). This is especially clear when compared with the levels of protein present in a cdc42-1 mutant, which shows drastically reduced amounts of the protein at all temperatures, consistent with previous analyses, but which grows normally at permissive temperature. Indirect immunofluorescence analysis of CDC42, cdc42-1, and cdc42-6 strains after a 1-h shift to restrictive temperature (33°C) indicated that unlike Cdc42-1 which was only detectable in 15% of small-budded cells, Cdc42-6 was clearly polarized to the bud tip in 78% of small-budded cells, virtually the same as wild-type Cdc42 (Fig. 8 B). We conclude that the phenotype of the cdc42-6 cells is unlikely to be a result of mislocalization of the mutant Cdc42-6 protein, but rather an inability of the mutant protein to carry out some other function.

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