Mmd1p, a novel, conserved protein essential for normal mitochondrial morphology and distribution in the fission yeast Schizosaccharomyces pombe

The mmd1 mutation causes temperature-sensitive growth and defects in mitochondrial morphology and distribution in the fission yeast Schizosaccharomyces pombe. In mutant cells, mitochondria aggregate at the two cell ends, with increased aggregation at elevated temperatures. Microtubules, which mediate mitochondrial positioning in fission yeast, seem normal in mmd1 cells at permissive temperature and after several hours at the nonpermissive temperature but display aberrant organization after prolonged periods at 37 degrees C. Additionally, cells harboring both mmd1 and ban5-4, a temperature-sensitive allele of alpha2-tubulin, display synthetic defects in growth and mitochondrial distribution. The mmd1 mutation maps to an open reading frame encoding a novel 35.7-kDa protein. The Mmd1p sequence features repeating EZ-HEAT motifs and displays high conservation with uncharacterized homologues found in a variety of organisms. Saccharomyces cerevisiae cells depleted for their MMD1 homologue show increased sensitivity to the antimicrotubule drug benomyl, and the S. cerevisiae gene complemented the S. pombe mutation. Mmd1p was localized to the cytosol. Mmd1p is the first identified component required for the alignment of mitochondria along microtubules in fission yeast.     

PXA domain-containing protein Pxa1 is required for normal vacuole function and morphology in Schizosaccharomyces pombe

PhoX homology (PX) domain-containing proteins play critical roles in vesicular trafficking, protein sorting, and lipid modification in eukaryotic cells. Several proteins with PX domains contain an associated domain termed PXA (PX-associated). Although PXA domain-containing proteins are required for some important cellular processes, the function of the PXA domain is unknown. We identified three PXA domain-containing proteins in Schizosaccharomyces pombe. S. pombe Pxa1p (SPAC5D6.07c) contained only the PXA domain, not the PX domain. To elucidate the role of the PXA domain in eukaryotic cells, we constructed and characterized a disruption mutant, pxa1. The pxa1 disruptant contained enlarged vacuoles and exhibited mislocalization of vacuolar carboxypeptidase Y (CPY). The conversion rate from pro- to mature-CPY was greatly impaired in pxa1 cells, and fluorescence microscopy indicated that a sorting receptor for CPY, Vps10p, mislocalized to the vacuolar membrane. The mutants were also deficient in vacuolar sorting of a multivesicular body (MVB) marker, a ubiquitin-GFP-carboxypeptidase S (Ub-GFP-CPS) fusion protein. Taken together, these results indicate that Pxa1 protein is required for normal vacuole function and morphology in S. pombe.     

The p21-activated kinase, Shk1, is required for proper regulation of microtubule dynamics in the fission yeast, Schizosaccharomyces pombe

The p21-activated kinase, Shk1, is required for the proper establishment of cell polarity in the fission yeast, Schizosaccharomyces pombe. We showed recently that loss of the essential Shk1 inhibitor, Skb15, causes significant spindle defects in fission yeast, thus implicating Shk1 as a potential regulator of microtubule dynamics. Here, we show that cells deficient in Shk1 function have malformed interphase microtubules and mitotic microtubule spindles, are hypersensitive to the microtubule-destabilizing drug thiabendazole (TBZ) and cold sensitive for growth. TBZ treatment causes a downregulation of Shk1 kinase activity, which increases rapidly after release of cells from the drug, thus providing a correlation between Shk1 kinase function and active microtubule polymerization. Consistent with a role for Shk1 as a regulator of microtubule dynamics, green fluorescent protein (GFP)-Shk1 fusion proteins localize to interphase microtubules and mitotic microtubule spindles, as well as to cell ends and septum-forming regions of fission yeast cells. We show that loss of Tea1, a cell end- and microtubule-localized protein previously implicated as a regulator of microtubule dynamics in fission yeast, exacerbates the growth and microtubule defects resulting from partial loss of Shk1 and that Shk1 localizes to illicit growth tips produced by tea1 mutant cells. Our results demonstrate that Shk1 is required for the proper regulation of microtubule dynamics in fission yeast and implicate Tea1 as a potential Shk1 regulator.     

The alpha-glucanase Agn1p is required for cell separation in Schizosaccharomyces pombe

BACKGROUND INFORMATION: In animal cells, cytokinesis occurs by constriction of an actomyosin ring. In fission yeast, ring constriction is followed by deposition of a multilayered division septum that must be cleaved to release the two daughter cells. Although many studies have focused on the actomyosin ring and septum assembly, little is known about the later steps involving the cleavage of the cell wall. RESULTS: We identified a novel gene in Schizosaccharomyces pombe, namely the agn1(+) gene that has homology to fungal 1,3-alpha-glucanases (mutanases). Disruption of the agn1(+) gene is not lethal to the cells, but does interfere with their separation, whereas overexpression of Agn1p is toxic and causes cell lysis. Agn1p levels reach a peak during septation and the protein localizes to the septum region before cell separation. Moreover, agn1(+) is responsible for the 1,3-alpha-glucanase activity, which shows a maximum at the end of septation. CONCLUSIONS: Our results clearly suggest the existence of a relationship between agn1(+), 1,3-alpha-glucanase activity and the completion of septation in S. pombe. Agn1p could be involved in the cleavage of the cylinder of the old wall that surrounds the primary septum, a region rich in alpha-glucans.     

Programmed cell death in fission yeast Schizosaccharomyces pombe

Yeasts have proven to be invaluable, genetically tractable systems to study various fundamental biological processes including programmed cell death. Recent advances in the elucidation of the molecular pathways underlying apoptotic cell death in yeasts have revealed remarkable similarities to mammalian apoptosis at cellular, organelle and macromolecular levels, thus making a strong case for the relevance of yeast models of regulated cell death. Programmed cell death has been reported in fission yeast Schizosaccharomyces pombe, primarily in the contexts of perturbed intracellular lipid metabolism, defective DNA replication, improper mitotic entry, chronological and replicative aging. Here we review the current understanding of the programmed cell death in fission yeast, paying particular attention to lipid-induced cell death. We discuss our recent findings that fission yeast exhibits plasticity of apoptotic and non-apoptotic modes of cell death in response to different lipid stimuli and growth conditions, and that mitochondria, reactive oxygen species and novel cell death mediators including metacaspase Pca1, SpRad9 and Pck1 are involved in the lipotoxic cell death. We also present perspectives on how various aspects of the cell and molecular biology of this organism can be explored to shed light on the governing principles underlying lipid-mediated signaling and cell demise.     

Dikaryotic cell division of the fission yeast Schizosaccharomyces pombe

Dikaryons, cells with two haploid nuclei contributed by the members of a mating pair, are part of the life cycle of many filamentous fungi, but the molecular mechanisms underlying the division of dikaryons are largely unknown. We found that the fission yeast Schizosaccharomyces pombe has a latent ability to divide as a dikaryon. Cells capable of restarting the mitotic cycle with two nuclei were prepared by transient inactivation of the septation initiation network. Close pairing of the two nuclei before mitosis was dependent on minus-end-directed kinesin Klp2p and was essential for propagation as a dikaryon. The two spindles extended in opposite directions, keeping their old spindle pole bodies at the prospective site of cell division until the mid-anaphase. The spindles then overlapped, exchanging the inner nuclei. Finally, twin mitosis was followed by a single cytokinesis, producing two daughter dikaryons carrying copies of the original pair of nuclei.     

DNA repair in the fission yeast, Schizosaccharomyces pombe

Mutants of the fission yeast Schizosaccharomyces pombe which are sensitive to UV and/or γ-irradiation have been assigned to 23 complementation groups, which can be assigned to three phenotypic groups. We have cloned genes which correct the deficiency in mutants corresponding to 12 of the complementation groups. Three genes in the excision-repair pathway have a high degree of sequence conservation with excision-repair genes from the evolutionarily distant budding yeast Saccharomyces cerevisiae. In contrast, those genes in the recombination repair pathway which have been characterised so far, show little homology with any previously characterised genes.     

Pheromone communication in the fission yeast Schizosaccharomyces pombe

Conjugation between two haploid yeast cells is generally controlled by the reciprocal action of diffusible mating pheromones, cells of each mating type releasing pheromones that induce mating-specific changes in cells of the opposite type. Recent studies into pheromone signalling in the fission yeast Schizosaccharomyces pombe have revealed significant parallels with processes in higher eukaryotes and could provide the opportunity for investigating communication in an organism that is amenable to both biochemical and genetic manipulation.     

Controlling cell cycle progress in the fission yeast Schizosaccharomyces pombe.

The suitability of fission yeast as a model for understanding the eukaryotic cell cycle has been validated in five years of exciting developments. We review recent advances in understanding the nature of the controls that regulate progression through the cell cycle and the coordination of DNA replication and mitosis.     

Featured organism: Schizosaccharomyces pombe, the fission yeast

Schizosaccharomyces pombe, the fission yeast, has long been a crucial model for the study of the eukaryote cell cycle. We take a look at this important yeast, whose genome has recently been completed, featuring comments from Valerie Wood, Jürg Bähler, Ramsay McFarlane, Susan Forsburg, Iain Hagan and Paul Nurse on the implications of having the complete sequence and future prospects for pombe genomics.     

Acetate assimilation by the fission yeast, Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe utilizes acetate at subinhibitory concentrations in the presence of D-glucose. The nonionized form of acetate is preferentially utilized, oxidized to 14CO2, and assimilated into lipids and proteins. Acetyl CoA synthetase activity greatly increases in the yeast cells grown in media containing acetate. However, glyoxylate cycle enzymes are not detectable in Schizosaccharomyces pombe. [1-14C]Acetate is incorporated into stereols, sterol esters, neutral lipids, and phospholipids. Assimilation of [1-14C]acetate into the peptide structure of proteins was confirmed by a proteolytic digestion experiment.