Characterization of vps33+, a gene required for vacuolar biogenesis and protein sorting in Schizosaccharomyces pombe

From the fission yeast Schizosaccharomyces pombe we have identified and deleted vps33, a gene encoding a homologue of VPS33, which is required for vacuolar biogenesis in S. cerevisiae cells. When the vps33(+) gene is disrupted, Sz. pombe strains are temperature-sensitive for growth and contain numerous small vesicular structures stained with FM4-64 in the cells. Deletion of the Sz. pombe vps33(+) gene results in pleiotropic phenotypes consistent with the absence of normal vacuoles, including missorting of vacuolar carboxypeptidase Y, various ion- and drug-sensitivities, and sporulation defects. These results are consistent with Vps33p being necessary for the morphogenesis of vacuoles and subsequent expression of vacuolar functions in Sz. pombe cells.     

Schizosaccharomyces pombe Pep12p is required for vacuolar protein transport and vacuolar homotypic fusion

In eukaryotic cells, SNARE proteins are essential for intracellular vesicle trafficking. Several SNARE proteins are required for vacuolar protein transport and vacuolar biogenesis in Saccharomyces cerevisiae. Previously we demonstrated that one of the fission yeast SNARE proteins, Pep12p, is not required for vacuolar fusion process in Schizosaccharomyces pombe. We have re-examined the function of S. pombe Pep12p using the newly created pep12(+) deletion strain. Deletion of the fission yeast pep12(+) gene results in pleiotropic phenotypes consistent with the absence of normal vacuoles, including missorting of vacuolar carboxypeptidase Y-and various ion- and drug-sensitivities. GFP-Pep12 fusion protein is mostly localized at the vacuolar membrane and the prevacuolar compartment. The S. pombe pep12Δ mutation phenocopies that of vps33Δ, suggesting that both Pep12p and Vps33p act at the same membrane fusion step in S. pombe, and both mutations cause vacuolar deficiency.     

Functional characterization of Gms1p/UDP-galactose transporter in Schizosaccharomyces pombe

Galactosylation of glycoproteins in the fission yeast Schizosaccharomyces pombe requires the transport of UDP-galactose as substrate for the galactosyltransferase into the lumen of the Golgi apparatus, which is achieved by the UDP-galactose transporter. We isolated a mutant (gms1) that is deficient in galactosylation of cell surface glycoproteins in Sz.pombe, and found that the gms1(+) gene encodes a UDP-galactose transporter. In the prediction of secondary structure of the Gms1 protein, an eight-membrane-spanning structure was obtained. Fluorescent microscopy revealed the functional Gms1-GFP fusion protein to be stably localized at the Golgi membrane. Sequencing analysis of the coding region of Gms1p derived from galactosylation-defective mutants identified a single amino acid mutation (A102T or A258E) located within the putative transmembrane region, helix 2 or helix 7, respectively. The mutagenized Gms1(A102T or A258E)p exhibited loss of UDP-galactose transport activity but no change in the localization to the Golgi membrane. The C-terminal truncated Gms1p mutants demonstrated that the C-terminal hydrophilic region was dispensable for targeting and function as UDP-galactose transporter at the Golgi membrane.We suggest that the putative eighth (the most C-terminus-proximal) transmembrane helix of Gms1p is critical to targeting from ER to the Golgi membrane.     

Construction of fission yeast vectors with a novel selection strategy that allows their use in wild-type fission yeasts

Novel vectors that use the Pichia pastoris INO1 gene as a selectable marker and exploit the natural inositol auxotrophy of the fission yeast are described. These plasmids also contained other features desirable in a plasmid cloning vector. These plasmids were evaluated in other species of Schizosaccharomyces and found to replicate autonomously in another variety of S. pombe, S. pombe var. malidevorans. These plasmids can be used for transformation of any wild-type S. pombe strain without the need for selection by induced auxotrophic mutations, or by selection by drug resistance markers, and should greatly assist genetic and molecular manipulations in these yeasts.     

Architectural features of pre-mRNA introns in the fission yeast Schizosaccharomyces pombe.

The architectural features of 73 introns found in 36 genes of the fission yeast Schizosaccharomyces pombe have been compiled and tabulated. The introns from S. pombe can be grouped into two size classes. Intron features are discussed in comparison to intron features of Saccharomyces cerevisiae and other eukaryotes. The results indicate that S. pombe displays quite different architectural features than the budding yeast S. cerevisiae. However, particularly in the 3' region, S. pombe introns also appear to differ from mammalian introns.     

Gene Ontology annotation status of the fission yeast genome: preliminary coverage approaches 100%

In this review, we present an overview of the Gene Ontology (GO) structure and describe how the GO is implemented for Sz. pombe and made available via Sz. pombe GeneDB (http://www.genedb.org/genedb/pombe/). We give a detailed progress report of Sz. pombe GO annotation, providing the current status of both manual and automatic annotations. Fission yeast has at least one GO annotation for 98.3% of its genes (excluding annotations to 'unknown' terms), greater than the current percentage coverage for any other organism. Approximately 65% (3225 gene products) have at least one annotation to each of the three ontologies (biological process, cellular component and molecular function). Approximately 30% (1443 gene products) have GO terms derived directly from small-scale experiments in fission yeast, supporting the validity of fission yeast as a model eukaryote and a reference organism.     

Characterization and behaviour of alpha-glucan synthase in Schizosaccharomyces pombe as revealed by electron microscopy

Alpha-1,3-Glucan is a cell wall component in Schizosaccharomyces pombe and is exclusive to budding yeast. We analysed the ultrastructure of the cell wall in the alpha-glucan synthase mutant mok1 and determined the role of alpha-1,3-glucan in cell wall formation of Sz. pombe. The mok1 mutant cell has an abnormal shape, with swelling at the tip or at the site of the septum. The cell wall is thicker and looser than that of wild-type cells, and the layered structure of the cell wall is broken. The glucan fibrils forming the protoplast retain a fine fibril structure, although their development into bundles is abnormal. We also report the localization of Mok1p by immunoelectron microscopy using high-pressure freeze substitution and SDS-digested freeze-fracture replica labelling methods. The Mok1p is localized on the cell membrane and moves from the cell tip to the medial region during the cell cycle. These results confirm that Mok1p plays an important role in the normal construction of the cell wall and in the primary step of glucan bundle formation, and that it is required for new cell wall synthesis during vegetative growth. These findings suggest that alpha-1,3-glucan is an essential component for cell wall formation in fission yeast.     

Acetate-glycerol cometabolism: cultivating Schizosaccharomyces pombe on a non-fermentable carbon source in a defined minimal medium

The growth of the fission yeast Schizosaccharomyces pombe on glucose and glycerol was monitored on-line in shake flasks and microtiter plates. The Edinburgh Minimal Medium 2 was improved by doubling its concentrations, improving its buffer and increasing its sulphur and iron concentrations additionally. By growing S. pombe on mixed carbon sources, it was shown that glycerol and glucose complement one another. Several tests were performed to establish the cultivation of S. pombe with non-fermentable glycerol as the main carbon source in minimal medium. Interestingly, a synergistic effect of glycerol and acetate was discovered which can significantly improve the growth of the fission yeast on glycerol. S. pombe showed optimal respiration activity, growth, and product formation by co-utilizing 20g/L glycerol and 2.5g/L sodium acetate.     

Schizosaccharomyces pombe Pmf1p is structurally and functionally related to Mmf1p of Saccharomyces cerevisiae

A novel family of small proteins, termed p14.5 or YERO57c/YJGFc, has been identified. Independent studies indicate that p14.5 family members are multifunctional proteins involved in several pathways, e.g. regulation of translation or activation of the protease mu-calpain. We have previously shown that Mmf1p, a p14.5 of the budding yeast Saccharomyces cerevisiae, is localized in the mitochondria and influences mitochondrial DNA stability. In addition, we have demonstrated that Mmf1p is functionally related to p14.5 of mammalian cells. To explore further the evolutionary conservation of the mitochondrial function(s) of the p14.5s we have extended our study to the fission yeast, Schizosaccharomyces pombe. In this organism two p14.5 homologous proteins are present: Pmf1p (pombe mitochondrial factor 1) and Hpm1p (homologous Pmf1p factor 1). We have generated a specific Pmf1p antibody, which recognizes a single band of approximately 15 kDa in total cellular extracts. Cellular fractionation experiments indicate that Pmf1p localizes in the mitochondria as well as in the cytoplasm. We also show that Pmf1p shares several properties of S. cerevisiae Mmf1p. Indeed, Pmf1p restores the wild-type phenotype when expressed in delta mmf1 S. cerevisiae cells. Deletion of the leader sequence of Pmf1p abrogates its ability to localize in mitochondria and to functionally replace Mmf1p. Thus, these data together with our previous study show that the mitochondrial function(s) of the p14.5 family members are highly conserved in eukaryotic cells.     

A fast and inexpensive method for random spore analysis in Schizosaccharomyces pombe

Random spore analysis is a fundamental tool of yeast genetics for determining gene linkage and the generation of recombinant progeny by genetic crosses. Experimentally it involves treatment of a mating mix with enzymes, such as zymolyase or lyticase, that selectively lyse the cell wall of vegetative cells rather than the spores. Here, we describe a method whereby the relative refractory nature of the spores to treatment with elevated temperature and repeated freeze-thawing facilitates random spore analysis at low cost in fission yeast Schizosaccharomyces pombe. Because of similar properties of spores in budding yeast, this method should prove to be useful for random spore analysis in both budding and fission yeasts.     

Isolation and characterization of Schizosaccharomyces pombe mutants lacking aminopeptidase activity

A mutant strain of the fission yeast Schizosaccharomyces pombe defective in aminopeptidase I was isolated by screening for lack of activity against the chromogenic substrate lysine-beta-naphthylamide in isolated colonies. Tetrad dissection of sporulated diploids heterozygous for the wild-type and mutant allele resulted in a 2:2 segregation of mutant and wild-type phenotype indicating a single chromosomal gene mutation. Gene dosage experiments indicated that the mutation might reside in the structural gene of aminopeptidase I. No vital consequences of aminopeptidase I deficiency on cell life and sporulation could be detected. However, the enzyme seems to be involved in protein degradation under conditions of nutrient deprivation.     

Schizosaccharomyces pombe Arc3 is a conserved subunit of the Arp2/3 complex required for polarity, actin organization, and endocytosis

We characterized the Schizosaccharomyces pombe arc3 gene, whose product shares sequence homology with that of the budding yeast ARC18 and human ARPC3/p21 subunits of the Arp2/3 complex. Our data showed that Arc3p co-localizes with F-actin patches at the cell ends, but not with F-actin cables or the equatorial actin ring, and binds other subunits of the Arp2/3 complex. Gene deletion analysis showed that arc3 is essential for viability. When arc3 expression was repressed, F-actin patches became dispersed throughout the cell with greatly reduced mobility. Furthermore, in arc3-repressed cells, endocytosis was also inhibited. Human ARPC3 rescued the viability of the Sz. pombe arc3 null mutant; in addition, ARPC3 also localized to F-actin patches in human cells. These data suggest that Arc3p is an evolutionarily conserved subunit of the Arp2/3 complex required for proper F-actin organization and efficient endocytosis.     

The deletion of CaVPS34 in the human pathogenic yeast Candida albicans causes defects in vesicle-mediated protein sorting and nuclear segregation

A Candida albicans null mutant of the phosphatidylinositol (PI) 3-kinase gene (CaVPS34) involved in virulence was examined by different microscopical techniques. We observed that vacuoles of the Cavps34 null mutant were considerably enlarged and electron-transparent. An interesting result obtained by transmission electron microscopy analysis of Cavps34 mutant cells was the aberrant patch-like accumulation of vesicles, which were localized in the periplasm close to the plasma membrane. We assume that the vesicles result from missorted prevacuolar compartments. In contrast to the accumulations of the specific endocytic dye FM4-64 in the vacuole membrane in C. albicans wild-type strains (ring staining pattern), the Cavps34 mutant strain showed a staining of punctuate structures, possibly multivesicular bodies (MVB), that are scattered all over the cell. This defect indicates a late block in endocytic vesicle transport. Measurement of the total activity of carboxypeptidase Y revealed significantly lower activity in Cavps34 mutant cells. This may indicate that carboxypeptidase Y is not properly activated as a result of mislocalization due to the lack of Vps34p. The deletion of the CaVPS34 gene caused disturbance of normal nuclear migration, which suggests that in the Cavps34 mutant the cell-size mediated control process of cell division is affected.     

A rapid and simple procedure for high-efficiency lithium acetate transformation of cryopreserved Schizosaccharomyces pombe cells

A rapid, simple, convenient, and highly efficient transformation of the fission yeast Schizosaccharomyces pombe has been developed. Freezing fission yeast cells in glycerol, a permeating cryoprotectant, with lithium acetate improved remarkably the transformation efficiency by one to two orders of magnitude. The optimum concentration of glycerol was found to be 30%, which is higher than that (10-15%) in the conventional cryopreservation of yeast cells. Glycerol not only played a role in cryopreserving the competent cells but also improved the transformation efficiency of the process. The thawed cell suspension with glycerol and lithium acetate was immediately mixed with carrier DNA, plasmid DNA and polyethylene glycol. Next, the mixture was heat shocked and directly spread on a selection plate. This simple procedure yielded more than 10(6) transformants/microg plasmid DNA, reducing the time required to only 20 min in total, including the thawing time. Furthermore, the frozen competent cells were stored long-term for more than 3 months without any significant loss of efficiency.     

Basic methods for fission yeast

The fission yeast Schizosaccharomyces pombe is a popular model system, and has been particularly influential in studies of the cell cycle and chromosome dynamics. Despite its differences from Saccharomyces cerevisiae, the tools and methods for fission yeast are conceptually similar to those used in budding yeast. Here, we present basic methods sufficient for a beginner in this system to carry out most required manipulations for genetic analysis or molecular biology.     

Mutational analysis of Schizosaccharomyces pombe U4 snRNA by plasmid exchange.

We have developed a system for testing mutations by plasmid exchange in the fission yeast Schizosaccharomyces pombe. This system has been used to test the requirement for different regions of the small nuclear RNA U4 in S. pombe. Surprisingly, five of seven deletion and substitution mutations tested in different regions of U4 prevent the accumulation of the mutant RNA. Substitution of the U4 sequence in stem 1 of the U4/U6 interaction domain allows accumulation of the mutant U4, but does not support viability. Two sequences with homology to the Sm binding site are found in the 3' region of S. pombe U4; substitution of the 3' sequence of the two does not interfere with accumulation or function of U4, indicating that the 5' sequence is the functional Sm-binding site.