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Exo-endocytic trafficking and the septin-based diffusion barrier are required for the maintenance of Cdc42p polarization during budding yeast asymmetric growth.

Orlando K, Sun X, Zhang J, Lu T, Yokomizo L, Wang P, Guo W - Mol. Biol. Cell (2011)

Bottom Line: We found that two separate exocytic pathways mediate Cdc42p delivery to the daughter cell.Defects in one of these pathways result in Cdc42p being rerouted through the other.A barrier function conferred by septins is required to counteract the dispersal of Cdc42p and maintain its localization in the daughter cell but has no effect on the initial polarization of Cdc42p at the presumptive budding site before symmetry breaking.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Pennsylvania, Philadelphia, PA 19096 College of Life Sciences, Nankai University, Tianjin, 300071, China.

ABSTRACT
Cdc42p plays a central role in asymmetric cell growth in yeast by controlling actin organization and vesicular trafficking. However, how Cdc42p is maintained specifically at the daughter cell plasma membrane during asymmetric cell growth is unclear. We have analyzed Cdc42p localization in yeast mutants defective in various stages of membrane trafficking by fluorescence microscopy and biochemical fractionation. We found that two separate exocytic pathways mediate Cdc42p delivery to the daughter cell. Defects in one of these pathways result in Cdc42p being rerouted through the other. In particular, the pathway involving trafficking through endosomes may couple Cdc42p endocytosis from, and subsequent redelivery to, the plasma membrane to maintain Cdc42p polarization at the daughter cell. Although the endo-exocytotic coupling is necessary for Cdc42p polarization, it is not sufficient to prevent the lateral diffusion of Cdc42p along the cell cortex. A barrier function conferred by septins is required to counteract the dispersal of Cdc42p and maintain its localization in the daughter cell but has no effect on the initial polarization of Cdc42p at the presumptive budding site before symmetry breaking. Collectively, membrane trafficking and septins function synergistically to maintain the dynamic polarization of Cdc42p during asymmetric growth in yeast.

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Analysis of Cdc42p association with different classes of secretory vesicles by Percoll density gradients. The sec10-2 mutant cells, which have block in both Bgl2p and invertase secretion, were used to analyze Cdc42p association with vesicles. A sec10-2 mutant strain with a copy of HA-Cdc42p integrated at its URA3 locus on the chromosome was used for membrane preparation as described in Materials and Methods. The secretory vesicle preparation was then subjected to Percoll density gradients. The collected fractions were analyzed by Western blot to measure the distributions of HA-Cdc42p (A) and Bgl2p (B) or tested for invertase activity (C). Two major peaks in the HA-Cdc42p blot were observed: one around fractions 5–9, which corresponds to the lighter Bgl2p vesicles, and the other around fractions 14–17, which corresponds to denser invertase vesicles.
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Figure 4: Analysis of Cdc42p association with different classes of secretory vesicles by Percoll density gradients. The sec10-2 mutant cells, which have block in both Bgl2p and invertase secretion, were used to analyze Cdc42p association with vesicles. A sec10-2 mutant strain with a copy of HA-Cdc42p integrated at its URA3 locus on the chromosome was used for membrane preparation as described in Materials and Methods. The secretory vesicle preparation was then subjected to Percoll density gradients. The collected fractions were analyzed by Western blot to measure the distributions of HA-Cdc42p (A) and Bgl2p (B) or tested for invertase activity (C). Two major peaks in the HA-Cdc42p blot were observed: one around fractions 5–9, which corresponds to the lighter Bgl2p vesicles, and the other around fractions 14–17, which corresponds to denser invertase vesicles.

Mentions: Invertase vesicles and Bgl2p vesicles have different densities (Harsay and Bretscher, 1995). Thus we examined the distribution of Cdc42p in these vesicles using density gradients. Secretory vesicle preparations from sec10-2, a mutant with defects in both Bgl2p and invertase secretion (He et al., 2007), were subjected to Percoll density gradients. The collected fractions were tested for invertase enzymatic activity and analyzed by Western blot for the distributions of Bgl2p and Cdc42p. Two major peaks in the HA-Cdc42p blot were observed (Figure 4A), one around fractions 5-9, which comigrate with the lighter Bgl2 vesicles (Figure 4B), and the other around fractions 14-17, which correspond to invertase activity (Figure 4C). The amount of Cdc42p in the lighter fractions was greater than that in the denser fractions. It is known that the majority of the exocytic vesicles are the Bgl2p-containing light-density vesicles, and the invertase vesicles constitute a small percentage of the total vesicles (Harsay and Bretscher, 1995). Therefore, we cannot infer from these results that the majority of Cdc42p are carried via the Bgl2p vesicles. The cofractionation of Cdc42p with both invertase activity and Bgl2p indicates that both vesicle populations are involved in the transport of Cdc42p to the plasma membrane. This result is also consistent with the fluorescence microscopy data above (Figures 1–3).


Exo-endocytic trafficking and the septin-based diffusion barrier are required for the maintenance of Cdc42p polarization during budding yeast asymmetric growth.

Orlando K, Sun X, Zhang J, Lu T, Yokomizo L, Wang P, Guo W - Mol. Biol. Cell (2011)

Analysis of Cdc42p association with different classes of secretory vesicles by Percoll density gradients. The sec10-2 mutant cells, which have block in both Bgl2p and invertase secretion, were used to analyze Cdc42p association with vesicles. A sec10-2 mutant strain with a copy of HA-Cdc42p integrated at its URA3 locus on the chromosome was used for membrane preparation as described in Materials and Methods. The secretory vesicle preparation was then subjected to Percoll density gradients. The collected fractions were analyzed by Western blot to measure the distributions of HA-Cdc42p (A) and Bgl2p (B) or tested for invertase activity (C). Two major peaks in the HA-Cdc42p blot were observed: one around fractions 5–9, which corresponds to the lighter Bgl2p vesicles, and the other around fractions 14–17, which corresponds to denser invertase vesicles.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046059&req=5

Figure 4: Analysis of Cdc42p association with different classes of secretory vesicles by Percoll density gradients. The sec10-2 mutant cells, which have block in both Bgl2p and invertase secretion, were used to analyze Cdc42p association with vesicles. A sec10-2 mutant strain with a copy of HA-Cdc42p integrated at its URA3 locus on the chromosome was used for membrane preparation as described in Materials and Methods. The secretory vesicle preparation was then subjected to Percoll density gradients. The collected fractions were analyzed by Western blot to measure the distributions of HA-Cdc42p (A) and Bgl2p (B) or tested for invertase activity (C). Two major peaks in the HA-Cdc42p blot were observed: one around fractions 5–9, which corresponds to the lighter Bgl2p vesicles, and the other around fractions 14–17, which corresponds to denser invertase vesicles.
Mentions: Invertase vesicles and Bgl2p vesicles have different densities (Harsay and Bretscher, 1995). Thus we examined the distribution of Cdc42p in these vesicles using density gradients. Secretory vesicle preparations from sec10-2, a mutant with defects in both Bgl2p and invertase secretion (He et al., 2007), were subjected to Percoll density gradients. The collected fractions were tested for invertase enzymatic activity and analyzed by Western blot for the distributions of Bgl2p and Cdc42p. Two major peaks in the HA-Cdc42p blot were observed (Figure 4A), one around fractions 5-9, which comigrate with the lighter Bgl2 vesicles (Figure 4B), and the other around fractions 14-17, which correspond to invertase activity (Figure 4C). The amount of Cdc42p in the lighter fractions was greater than that in the denser fractions. It is known that the majority of the exocytic vesicles are the Bgl2p-containing light-density vesicles, and the invertase vesicles constitute a small percentage of the total vesicles (Harsay and Bretscher, 1995). Therefore, we cannot infer from these results that the majority of Cdc42p are carried via the Bgl2p vesicles. The cofractionation of Cdc42p with both invertase activity and Bgl2p indicates that both vesicle populations are involved in the transport of Cdc42p to the plasma membrane. This result is also consistent with the fluorescence microscopy data above (Figures 1–3).

Bottom Line: We found that two separate exocytic pathways mediate Cdc42p delivery to the daughter cell.Defects in one of these pathways result in Cdc42p being rerouted through the other.A barrier function conferred by septins is required to counteract the dispersal of Cdc42p and maintain its localization in the daughter cell but has no effect on the initial polarization of Cdc42p at the presumptive budding site before symmetry breaking.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Pennsylvania, Philadelphia, PA 19096 College of Life Sciences, Nankai University, Tianjin, 300071, China.

ABSTRACT
Cdc42p plays a central role in asymmetric cell growth in yeast by controlling actin organization and vesicular trafficking. However, how Cdc42p is maintained specifically at the daughter cell plasma membrane during asymmetric cell growth is unclear. We have analyzed Cdc42p localization in yeast mutants defective in various stages of membrane trafficking by fluorescence microscopy and biochemical fractionation. We found that two separate exocytic pathways mediate Cdc42p delivery to the daughter cell. Defects in one of these pathways result in Cdc42p being rerouted through the other. In particular, the pathway involving trafficking through endosomes may couple Cdc42p endocytosis from, and subsequent redelivery to, the plasma membrane to maintain Cdc42p polarization at the daughter cell. Although the endo-exocytotic coupling is necessary for Cdc42p polarization, it is not sufficient to prevent the lateral diffusion of Cdc42p along the cell cortex. A barrier function conferred by septins is required to counteract the dispersal of Cdc42p and maintain its localization in the daughter cell but has no effect on the initial polarization of Cdc42p at the presumptive budding site before symmetry breaking. Collectively, membrane trafficking and septins function synergistically to maintain the dynamic polarization of Cdc42p during asymmetric growth in yeast.

Show MeSH
Related in: MedlinePlus