Search

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.

Microtubules and the protein tea1p have important roles in regulating cell polarity in the fission yeast Schizosaccharomyces pombe. Here, using combinations of drugs, environmental perturbations and genetic mutants, we demonstrate that once a cell polarity axis is established, microtubules have at best a minor role in maintaining the cortical actin cytoskeleton and the rate and direction of cell growth. In addition, we find that after perturbations that disrupt cell polarity and the cortical actin cytoskeleton, microtubules are not required for re-establishment of polarity per se. However, after such perturbations, the distribution of cytoplasmic microtubules plays an important role in dictating the position of sites of polarity re-establishment. Furthermore, this influence of microtubule distribution on site selection during polarity re-establishment requires the presence of tea1p, suggesting that tea1p is crucial for coupling microtubule distribution to the regulation of cell polarity. Our results suggest a model in which, at the cellular level, two distinct and separable mechanisms contribute to how tea1p regulates site selection during polarity re-establishment. First, tea1p remaining at cell tips after cortical depolarization can serve as a cortical landmark for microtubule-independent site selection; second, tea1p newly targeted to the cell cortex by association with microtubules can promote the formation of polarity axes de novo.

A Drosophila strain, carrying a P[lacW] element in the promoter of the protein phosphatase 2A (PP2A) catalytic subunit gene at chromosomal location 28D, has been identified using plasmid rescue of the P element and adjoining genomic DNA in Escherichia coli. Reversion mutagenesis was employed to demonstrate that the observed phenotype of the Drosophila strain was due to a single P[lacW] element insertion at 28D and to create three deficiency strains at this locus. Drosophila heterozygous for P[lacW]28D have reduced levels of PP2A mRNA and reduced PP2A catalytic activity against four different substrates compared to wild type, while homozygotes are deduced to have approximately 20% of wild-type PP2A activity. P[lacW]28D homozygotes, termed microtubule star (mts), die in embryo-genesis around the time of cellularisation, exhibiting over-condensed chromatin and a block in mitosis between prophase and the initiation of anaphase. Multiple centrosomes are visible in cellularised embryos, suggesting that PP2A may play a role in coupling the nuclear and centrosome cycles. When embryos arrest just prior to cellularisation, disorganised elongated arrays of microtubules radiate from centrosomes in all directions, but they are rarely associated with any DNA, suggesting that PP2A is required for the attachment of microtubules to chromosomal DNA at the kinetochore.