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Vesicle docking to the spindle pole body is necessary to recruit the exocyst during membrane formation in Saccharomyces cerevisiae.

Mathieson EM, Suda Y, Nickas M, Snydsman B, Davis TN, Muller EG, Neiman AM - Mol. Biol. Cell (2010)

Bottom Line: During meiosis II in Saccharomyces cerevisiae, the cytoplasmic face of the spindle pole body, referred to as the meiosis II outer plaque (MOP), is modified in both composition and structure to become the initiation site for de novo formation of a membrane called the prospore membrane.The N-termini of two proteins, Mpc54p and Spo21p, were oriented toward the outer surface of the structure.Mutations in the N-terminus of Mpc54p resulted in a unique phenotype: precursor vesicles loosely tethered to the MOP but did not contact its surface.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794-5215, USA.

ABSTRACT
During meiosis II in Saccharomyces cerevisiae, the cytoplasmic face of the spindle pole body, referred to as the meiosis II outer plaque (MOP), is modified in both composition and structure to become the initiation site for de novo formation of a membrane called the prospore membrane. The MOP serves as a docking complex for precursor vesicles that are targeted to its surface. Using fluorescence resonance energy transfer analysis, the orientation of coiled-coil proteins within the MOP has been determined. The N-termini of two proteins, Mpc54p and Spo21p, were oriented toward the outer surface of the structure. Mutations in the N-terminus of Mpc54p resulted in a unique phenotype: precursor vesicles loosely tethered to the MOP but did not contact its surface. Thus, these mpc54 mutants separate the steps of vesicle association and docking. Using these mpc54 mutants, we determined that recruitment of the Rab GTPase Sec4p, as well as the exocyst components Sec3p and Sec8p, to the precursor vesicles requires vesicle docking to the MOP. This suggests that the MOP promotes membrane formation both by localization of precursor vesicles to a particular site and by recruitment of a second tethering complex, the exocyst, that stimulates downstream events of fusion.

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The mpc54-RFP mutant alleles disrupt spore formation. (A) Sporulation efficiency of an mpc54Δ strain transformed with Mpc54p mutants under the MPC54 promoter was monitored using light microscopy. Values shown are the average of three experiments in which 200 cells were analyzed for sporulation in each experiment. Error bars, 1 SD. (B) The distribution of ascal types in an mpc54Δ strain transformed with Mpc54p mutants under the MPC54 promoter was monitored using light microscopy.
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Figure 3: The mpc54-RFP mutant alleles disrupt spore formation. (A) Sporulation efficiency of an mpc54Δ strain transformed with Mpc54p mutants under the MPC54 promoter was monitored using light microscopy. Values shown are the average of three experiments in which 200 cells were analyzed for sporulation in each experiment. Error bars, 1 SD. (B) The distribution of ascal types in an mpc54Δ strain transformed with Mpc54p mutants under the MPC54 promoter was monitored using light microscopy.

Mentions: If the mpc54 mutants disrupt the function of Mpc54p during prospore membrane formation, then transforming these alleles into an mpc54Δ strain would not rescue sporulation. All strains expressing the Mpc54p mutant proteins on a plasmid displayed a dramatic reduction or a complete loss of sporulation compared with wild-type Mpc54p-RFP (Figure 3A). Asci that did form in the mutants were monads or dyads, also indicating a defect in sporulation (Figure 3B). Assembly of the MOP structure is particularly susceptible to expression levels of the MOP components (Bajgier et al., 2001; Nickas et al., 2003). To ensure that the mutant phenotypes observed were not an indirect effect of reduced expression levels, the mutant alleles were placed on high-copy plasmids or were integrated into chromosomes. Under these different conditions the sporulation efficiency was similar for each mpc54 mutant (Supplementary Table 3), indicating that these phenotypes are due to changes in the functionality of the mutant forms of Mpc54p and not simply due to reduced expression of the protein. This suggests that the mutations in the different alleles directly disrupt spore formation.


Vesicle docking to the spindle pole body is necessary to recruit the exocyst during membrane formation in Saccharomyces cerevisiae.

Mathieson EM, Suda Y, Nickas M, Snydsman B, Davis TN, Muller EG, Neiman AM - Mol. Biol. Cell (2010)

The mpc54-RFP mutant alleles disrupt spore formation. (A) Sporulation efficiency of an mpc54Δ strain transformed with Mpc54p mutants under the MPC54 promoter was monitored using light microscopy. Values shown are the average of three experiments in which 200 cells were analyzed for sporulation in each experiment. Error bars, 1 SD. (B) The distribution of ascal types in an mpc54Δ strain transformed with Mpc54p mutants under the MPC54 promoter was monitored using light microscopy.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2965686&req=5

Figure 3: The mpc54-RFP mutant alleles disrupt spore formation. (A) Sporulation efficiency of an mpc54Δ strain transformed with Mpc54p mutants under the MPC54 promoter was monitored using light microscopy. Values shown are the average of three experiments in which 200 cells were analyzed for sporulation in each experiment. Error bars, 1 SD. (B) The distribution of ascal types in an mpc54Δ strain transformed with Mpc54p mutants under the MPC54 promoter was monitored using light microscopy.
Mentions: If the mpc54 mutants disrupt the function of Mpc54p during prospore membrane formation, then transforming these alleles into an mpc54Δ strain would not rescue sporulation. All strains expressing the Mpc54p mutant proteins on a plasmid displayed a dramatic reduction or a complete loss of sporulation compared with wild-type Mpc54p-RFP (Figure 3A). Asci that did form in the mutants were monads or dyads, also indicating a defect in sporulation (Figure 3B). Assembly of the MOP structure is particularly susceptible to expression levels of the MOP components (Bajgier et al., 2001; Nickas et al., 2003). To ensure that the mutant phenotypes observed were not an indirect effect of reduced expression levels, the mutant alleles were placed on high-copy plasmids or were integrated into chromosomes. Under these different conditions the sporulation efficiency was similar for each mpc54 mutant (Supplementary Table 3), indicating that these phenotypes are due to changes in the functionality of the mutant forms of Mpc54p and not simply due to reduced expression of the protein. This suggests that the mutations in the different alleles directly disrupt spore formation.

Bottom Line: During meiosis II in Saccharomyces cerevisiae, the cytoplasmic face of the spindle pole body, referred to as the meiosis II outer plaque (MOP), is modified in both composition and structure to become the initiation site for de novo formation of a membrane called the prospore membrane.The N-termini of two proteins, Mpc54p and Spo21p, were oriented toward the outer surface of the structure.Mutations in the N-terminus of Mpc54p resulted in a unique phenotype: precursor vesicles loosely tethered to the MOP but did not contact its surface.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794-5215, USA.

ABSTRACT
During meiosis II in Saccharomyces cerevisiae, the cytoplasmic face of the spindle pole body, referred to as the meiosis II outer plaque (MOP), is modified in both composition and structure to become the initiation site for de novo formation of a membrane called the prospore membrane. The MOP serves as a docking complex for precursor vesicles that are targeted to its surface. Using fluorescence resonance energy transfer analysis, the orientation of coiled-coil proteins within the MOP has been determined. The N-termini of two proteins, Mpc54p and Spo21p, were oriented toward the outer surface of the structure. Mutations in the N-terminus of Mpc54p resulted in a unique phenotype: precursor vesicles loosely tethered to the MOP but did not contact its surface. Thus, these mpc54 mutants separate the steps of vesicle association and docking. Using these mpc54 mutants, we determined that recruitment of the Rab GTPase Sec4p, as well as the exocyst components Sec3p and Sec8p, to the precursor vesicles requires vesicle docking to the MOP. This suggests that the MOP promotes membrane formation both by localization of precursor vesicles to a particular site and by recruitment of a second tethering complex, the exocyst, that stimulates downstream events of fusion.

Show MeSH
Related in: MedlinePlus