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Characterization of Mug33 reveals complementary roles for actin cable-dependent transport and exocyst regulators in fission yeast exocytosis.

Snaith HA, Thompson J, Yates JR, Sawin KE - J. Cell. Sci. (2011)

Bottom Line: Although mug33Δ mutants are viable, with only a mild cell-polarity phenotype, mug33Δ myo52Δ double mutants are synthetically lethal.Combining mug33 Δ with deletion of the formin For3 (for3Δ) leads to synthetic temperature-sensitive growth and strongly reduced levels of exocytosis.Interestingly, mutants in non-essential genes involved in exocyst function behave in a manner similar to mug33Δ when combined with myo52Δ and for3Δ.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Swann Building, Mayfield Road, Edinburgh EH93JR, UK.

ABSTRACT
Although endocytosis and exocytosis have been extensively studied in budding yeast, there have been relatively few investigations of these complex processes in the fission yeast Schizosaccharomyces pombe. Here we identify and characterize fission yeast Mug33, a novel Tea1-interacting protein, and show that Mug33 is involved in exocytosis. Mug33 is a Sur7/PalI-family transmembrane protein that localizes to the plasma membrane at the cell tips and to cytoplasmic tubulovesicular elements (TVEs). A subset of Mug33 TVEs make long-range movements along actin cables, co-translocating with subunits of the exocyst complex. TVE movement depends on the type V myosin Myo52. Although mug33Δ mutants are viable, with only a mild cell-polarity phenotype, mug33Δ myo52Δ double mutants are synthetically lethal. Combining mug33 Δ with deletion of the formin For3 (for3Δ) leads to synthetic temperature-sensitive growth and strongly reduced levels of exocytosis. Interestingly, mutants in non-essential genes involved in exocyst function behave in a manner similar to mug33Δ when combined with myo52Δ and for3Δ. By contrast, combining mug33Δ with mutants in non-essential exocyst genes has only minor effects on growth. We propose that Mug33 contributes to exocyst function and that actin cable-dependent vesicle transport and exocyst function have complementary roles in promoting efficient exocytosis in fission yeast.

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Mug33 colocalizes with components of the exocytic pathway. (A) Colocalization of Mug33–mKate (green) with GFP–Syb1 (red) in a wild-type cell. (B) Kymograph of a different wild-type cell, showing co-translocation of Mug33–mKate TVEs with GFP–Syb1. Kymograph presentation is as in Fig. 3; the total time is 96 seconds. (C) Time-lapse montage and associated kymograph showing co-translocation of Mug33–mKate (green) with Sec6–GFP (red) in a wild-type cell. Labelling as in Fig. 4E; the total time in kymograph is 36 seconds. (D) Time-lapse montage and associated kymograph showing co-translocation of Mug33–mKate (green) with Sec8–GFP (red) in a wild-type cell. Labeling is as in Fig. 4E; the total time in kymograph is 18 seconds. (E) Localization of Sec8–GFP in wild-type and mug33Δ cells. (F) Localization of Mug33–GFP in wild-type cells at 36°C, sec8-1 at 25°C and 36°C and rho3Δ at 36°C. (G) Anti-GFP antibody immunoblot of Mug33–GFP in sec8-1 at 25°C and 36°C. Scale bars: 5 μm.
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Figure 6: Mug33 colocalizes with components of the exocytic pathway. (A) Colocalization of Mug33–mKate (green) with GFP–Syb1 (red) in a wild-type cell. (B) Kymograph of a different wild-type cell, showing co-translocation of Mug33–mKate TVEs with GFP–Syb1. Kymograph presentation is as in Fig. 3; the total time is 96 seconds. (C) Time-lapse montage and associated kymograph showing co-translocation of Mug33–mKate (green) with Sec6–GFP (red) in a wild-type cell. Labelling as in Fig. 4E; the total time in kymograph is 36 seconds. (D) Time-lapse montage and associated kymograph showing co-translocation of Mug33–mKate (green) with Sec8–GFP (red) in a wild-type cell. Labeling is as in Fig. 4E; the total time in kymograph is 18 seconds. (E) Localization of Sec8–GFP in wild-type and mug33Δ cells. (F) Localization of Mug33–GFP in wild-type cells at 36°C, sec8-1 at 25°C and 36°C and rho3Δ at 36°C. (G) Anti-GFP antibody immunoblot of Mug33–GFP in sec8-1 at 25°C and 36°C. Scale bars: 5 μm.

Mentions: If Mug33 were cycling between TVEs and the plasma membrane, one would also expect Mug33 to be present in exocytic compartments. To investigate this we co-imaged Mug33 with Syb1, the fission yeast homolog of the vesicle SNAREs mammalian synaptobrevin and budding yeast Snc1p, which regulate fusion of exocytic vesicles with the plasma membrane (Baumert et al., 1989; Edamatsu and Toyoshima, 2003; Gurunathan et al., 2000). Mug33 and Syb1 were present in the same TVEs and co-translocated together (Fig. 6A,B). We also found that Mug33 partially colocalized with exocyst subunits Sec6 and Sec8 at cell tips, and some cytoplasmic Mug33 TVEs co-translocated with these proteins (Fig. 6C,D; supplementary material Movies 13, 14). Localization of Sec6, Sec8 and Sec10 was normal in mug33Δ cells (Fig. 6E; supplementary material Fig. S3A). However, growth of temperature-sensitive sec8-1 mutant cells at the restrictive temperature led to the disappearance of Mug33–GFP from cell tips and a reduction in TVEs, despite levels of Mug33–GFP being unaltered (Fig. 6F,G). Mug33 localization was also impaired in cells lacking the exocyst-activating GTPase Rho3 (Fig. 6F). These data indicate that Mug33 colocalizes with the exocyst on exocytic vesicles and that Mug33 depends on an intact exocyst complex for proper localization on TVEs and at cell tips.


Characterization of Mug33 reveals complementary roles for actin cable-dependent transport and exocyst regulators in fission yeast exocytosis.

Snaith HA, Thompson J, Yates JR, Sawin KE - J. Cell. Sci. (2011)

Mug33 colocalizes with components of the exocytic pathway. (A) Colocalization of Mug33–mKate (green) with GFP–Syb1 (red) in a wild-type cell. (B) Kymograph of a different wild-type cell, showing co-translocation of Mug33–mKate TVEs with GFP–Syb1. Kymograph presentation is as in Fig. 3; the total time is 96 seconds. (C) Time-lapse montage and associated kymograph showing co-translocation of Mug33–mKate (green) with Sec6–GFP (red) in a wild-type cell. Labelling as in Fig. 4E; the total time in kymograph is 36 seconds. (D) Time-lapse montage and associated kymograph showing co-translocation of Mug33–mKate (green) with Sec8–GFP (red) in a wild-type cell. Labeling is as in Fig. 4E; the total time in kymograph is 18 seconds. (E) Localization of Sec8–GFP in wild-type and mug33Δ cells. (F) Localization of Mug33–GFP in wild-type cells at 36°C, sec8-1 at 25°C and 36°C and rho3Δ at 36°C. (G) Anti-GFP antibody immunoblot of Mug33–GFP in sec8-1 at 25°C and 36°C. Scale bars: 5 μm.
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Figure 6: Mug33 colocalizes with components of the exocytic pathway. (A) Colocalization of Mug33–mKate (green) with GFP–Syb1 (red) in a wild-type cell. (B) Kymograph of a different wild-type cell, showing co-translocation of Mug33–mKate TVEs with GFP–Syb1. Kymograph presentation is as in Fig. 3; the total time is 96 seconds. (C) Time-lapse montage and associated kymograph showing co-translocation of Mug33–mKate (green) with Sec6–GFP (red) in a wild-type cell. Labelling as in Fig. 4E; the total time in kymograph is 36 seconds. (D) Time-lapse montage and associated kymograph showing co-translocation of Mug33–mKate (green) with Sec8–GFP (red) in a wild-type cell. Labeling is as in Fig. 4E; the total time in kymograph is 18 seconds. (E) Localization of Sec8–GFP in wild-type and mug33Δ cells. (F) Localization of Mug33–GFP in wild-type cells at 36°C, sec8-1 at 25°C and 36°C and rho3Δ at 36°C. (G) Anti-GFP antibody immunoblot of Mug33–GFP in sec8-1 at 25°C and 36°C. Scale bars: 5 μm.
Mentions: If Mug33 were cycling between TVEs and the plasma membrane, one would also expect Mug33 to be present in exocytic compartments. To investigate this we co-imaged Mug33 with Syb1, the fission yeast homolog of the vesicle SNAREs mammalian synaptobrevin and budding yeast Snc1p, which regulate fusion of exocytic vesicles with the plasma membrane (Baumert et al., 1989; Edamatsu and Toyoshima, 2003; Gurunathan et al., 2000). Mug33 and Syb1 were present in the same TVEs and co-translocated together (Fig. 6A,B). We also found that Mug33 partially colocalized with exocyst subunits Sec6 and Sec8 at cell tips, and some cytoplasmic Mug33 TVEs co-translocated with these proteins (Fig. 6C,D; supplementary material Movies 13, 14). Localization of Sec6, Sec8 and Sec10 was normal in mug33Δ cells (Fig. 6E; supplementary material Fig. S3A). However, growth of temperature-sensitive sec8-1 mutant cells at the restrictive temperature led to the disappearance of Mug33–GFP from cell tips and a reduction in TVEs, despite levels of Mug33–GFP being unaltered (Fig. 6F,G). Mug33 localization was also impaired in cells lacking the exocyst-activating GTPase Rho3 (Fig. 6F). These data indicate that Mug33 colocalizes with the exocyst on exocytic vesicles and that Mug33 depends on an intact exocyst complex for proper localization on TVEs and at cell tips.

Bottom Line: Although mug33Δ mutants are viable, with only a mild cell-polarity phenotype, mug33Δ myo52Δ double mutants are synthetically lethal.Combining mug33 Δ with deletion of the formin For3 (for3Δ) leads to synthetic temperature-sensitive growth and strongly reduced levels of exocytosis.Interestingly, mutants in non-essential genes involved in exocyst function behave in a manner similar to mug33Δ when combined with myo52Δ and for3Δ.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Swann Building, Mayfield Road, Edinburgh EH93JR, UK.

ABSTRACT
Although endocytosis and exocytosis have been extensively studied in budding yeast, there have been relatively few investigations of these complex processes in the fission yeast Schizosaccharomyces pombe. Here we identify and characterize fission yeast Mug33, a novel Tea1-interacting protein, and show that Mug33 is involved in exocytosis. Mug33 is a Sur7/PalI-family transmembrane protein that localizes to the plasma membrane at the cell tips and to cytoplasmic tubulovesicular elements (TVEs). A subset of Mug33 TVEs make long-range movements along actin cables, co-translocating with subunits of the exocyst complex. TVE movement depends on the type V myosin Myo52. Although mug33Δ mutants are viable, with only a mild cell-polarity phenotype, mug33Δ myo52Δ double mutants are synthetically lethal. Combining mug33 Δ with deletion of the formin For3 (for3Δ) leads to synthetic temperature-sensitive growth and strongly reduced levels of exocytosis. Interestingly, mutants in non-essential genes involved in exocyst function behave in a manner similar to mug33Δ when combined with myo52Δ and for3Δ. By contrast, combining mug33Δ with mutants in non-essential exocyst genes has only minor effects on growth. We propose that Mug33 contributes to exocyst function and that actin cable-dependent vesicle transport and exocyst function have complementary roles in promoting efficient exocytosis in fission yeast.

Show MeSH
Related in: MedlinePlus