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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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For3 and Myo52 are required for Mug33 translocation. (A) Localization of Mug33–GFP in for3Δ and myo52Δ cells and Mug33–mKate in myo51Δ cells. (B) Kymographs showing Mug33–GFP TVE translocations or absence of translocations in individual mutant cells. Kymograph presentation is as in Fig. 3; the total time is 36 seconds. (C,D) Mug33 and For3 do not co-translocate. Kymographs from single z-sections in two wild-type cells coexpressing Mug33–mKate (green) and For3-3xGFP (red); total time is 50 seconds. (E) Time-lapse montage and associated kymograph showing co-translocation of Mug33–mKate TVEs (green) with Myo52–GFP (red) in wild-type cells. Green and red arrowheads indicate the same TVE in different channels. The elapsed time is shown in seconds. Kymograph presentation is as in Fig. 3; the total time is 36 seconds. Arrowheads in the kymograph indicate first and last time-points of the montage. (F) Length and speed of Mug33 TVE translocations in untreated wild-type, tea1Δ, myo51Δ, for3Δ and myo52Δ cells and in wild-type cells treated with MBC and LatB. The different segments within each column indicate the translocation events within the indicated length or speed ranges. See also Table 1. Scale bars: 5 μm.
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Figure 4: For3 and Myo52 are required for Mug33 translocation. (A) Localization of Mug33–GFP in for3Δ and myo52Δ cells and Mug33–mKate in myo51Δ cells. (B) Kymographs showing Mug33–GFP TVE translocations or absence of translocations in individual mutant cells. Kymograph presentation is as in Fig. 3; the total time is 36 seconds. (C,D) Mug33 and For3 do not co-translocate. Kymographs from single z-sections in two wild-type cells coexpressing Mug33–mKate (green) and For3-3xGFP (red); total time is 50 seconds. (E) Time-lapse montage and associated kymograph showing co-translocation of Mug33–mKate TVEs (green) with Myo52–GFP (red) in wild-type cells. Green and red arrowheads indicate the same TVE in different channels. The elapsed time is shown in seconds. Kymograph presentation is as in Fig. 3; the total time is 36 seconds. Arrowheads in the kymograph indicate first and last time-points of the montage. (F) Length and speed of Mug33 TVE translocations in untreated wild-type, tea1Δ, myo51Δ, for3Δ and myo52Δ cells and in wild-type cells treated with MBC and LatB. The different segments within each column indicate the translocation events within the indicated length or speed ranges. See also Table 1. Scale bars: 5 μm.

Mentions: The fast movement of Mug33 TVEs persisted after disruption of microtubules with MBC but was abolished upon disruption of filamentous actin with latrunculin B (LatB; Fig. 3A,B; Fig. 4F) suggesting that translocation involves the actin cytoskeleton. Co-imaging Mug33–mKate with actin filaments (GFP–CHDRng2) showed that Mug33 TVEs associated with and translocated along actin cables, in both wild-type and tea1Δ cells (Fig. 3C,D, Fig. 4F; supplementary material Movies 3, 4).


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)

For3 and Myo52 are required for Mug33 translocation. (A) Localization of Mug33–GFP in for3Δ and myo52Δ cells and Mug33–mKate in myo51Δ cells. (B) Kymographs showing Mug33–GFP TVE translocations or absence of translocations in individual mutant cells. Kymograph presentation is as in Fig. 3; the total time is 36 seconds. (C,D) Mug33 and For3 do not co-translocate. Kymographs from single z-sections in two wild-type cells coexpressing Mug33–mKate (green) and For3-3xGFP (red); total time is 50 seconds. (E) Time-lapse montage and associated kymograph showing co-translocation of Mug33–mKate TVEs (green) with Myo52–GFP (red) in wild-type cells. Green and red arrowheads indicate the same TVE in different channels. The elapsed time is shown in seconds. Kymograph presentation is as in Fig. 3; the total time is 36 seconds. Arrowheads in the kymograph indicate first and last time-points of the montage. (F) Length and speed of Mug33 TVE translocations in untreated wild-type, tea1Δ, myo51Δ, for3Δ and myo52Δ cells and in wild-type cells treated with MBC and LatB. The different segments within each column indicate the translocation events within the indicated length or speed ranges. See also Table 1. Scale bars: 5 μm.
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Figure 4: For3 and Myo52 are required for Mug33 translocation. (A) Localization of Mug33–GFP in for3Δ and myo52Δ cells and Mug33–mKate in myo51Δ cells. (B) Kymographs showing Mug33–GFP TVE translocations or absence of translocations in individual mutant cells. Kymograph presentation is as in Fig. 3; the total time is 36 seconds. (C,D) Mug33 and For3 do not co-translocate. Kymographs from single z-sections in two wild-type cells coexpressing Mug33–mKate (green) and For3-3xGFP (red); total time is 50 seconds. (E) Time-lapse montage and associated kymograph showing co-translocation of Mug33–mKate TVEs (green) with Myo52–GFP (red) in wild-type cells. Green and red arrowheads indicate the same TVE in different channels. The elapsed time is shown in seconds. Kymograph presentation is as in Fig. 3; the total time is 36 seconds. Arrowheads in the kymograph indicate first and last time-points of the montage. (F) Length and speed of Mug33 TVE translocations in untreated wild-type, tea1Δ, myo51Δ, for3Δ and myo52Δ cells and in wild-type cells treated with MBC and LatB. The different segments within each column indicate the translocation events within the indicated length or speed ranges. See also Table 1. Scale bars: 5 μm.
Mentions: The fast movement of Mug33 TVEs persisted after disruption of microtubules with MBC but was abolished upon disruption of filamentous actin with latrunculin B (LatB; Fig. 3A,B; Fig. 4F) suggesting that translocation involves the actin cytoskeleton. Co-imaging Mug33–mKate with actin filaments (GFP–CHDRng2) showed that Mug33 TVEs associated with and translocated along actin cables, in both wild-type and tea1Δ cells (Fig. 3C,D, Fig. 4F; supplementary material Movies 3, 4).

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