Dielectric spectroscopy of Schizosaccharomyces pombe using electrorotation and electroorientation

Two complementary AC electrokinetic techniques electrorotation (ER) and electroorientation (EO) enabled the dielectric characterization of the rod-shaped fission yeast Schizosaccharomyces pombe. The use of microstructured electrodes allowed both ER and EO measurements to be performed over wide ranges of field frequency and medium conductivity. Due to their layered structure, living S. pombe cells exhibited up to three well resolved peaks in their ER spectra and also two distinct orientations, i.e., parallel or perpendicular to the imposed linear field. Heat treatment and enzymatic protoplast isolation led to dramatic changes in the electrokinetic behavior of fission yeast. Application of the theoretical models linking the ER and EO spectra yielded the dielectric parameters of the major structural units of S. pombe cells (cell wall, plasma membrane and cytosol). The dielectric characterization of yeasts has an enormous impact in biotechnology and biomedicine, because electric field pulse techniques (electrofusion and electropermeabilization) are widely used for production of transgenic yeast strains of economic importance. The present study also showed that combined ER and EO measurements can be employed as a powerful diagnostic tool for analyzing changes in yeast structure and physiology upon exposure to various stress conditions.     

Dielectric measurement of individual microtubules using the electroorientation method

Little is known about the electrostatic/dynamic properties of microtubules, which are considered to underlie their electrostatic interactions with various proteins such as motor proteins, microtubule-associated proteins, and microtubules themselves (lateral association of microtubules). To measure the dielectric properties of microtubules, we developed an experiment system in which the electroorientation of microtubules was observed under a dark-field microscope. Upon application of an alternating electric field (0.5-1.9 x 10(5) V/m, 10 kHz-3 MHz), the microtubules were oriented parallel to the field line in a few seconds because of the dipole moment induced along their long axes. The process of this orientation was analyzed based on a dielectric ellipsoid model, and the conductivity and dielectric constant of each microtubule were calculated. The analyses revealed that the microtubules were highly conductive, which is consistent with the counterion polarization model-counterions bound to highly negatively charged microtubules can move along the long axis, and this mobility might be the origin of the high conductivity. Our experiment system provides a useful tool to quantitatively evaluate the polyelectrolyte nature of microtubules, thus paving the way for future studies aiming to understand the physicochemical mechanism underlying the electrostatic interactions of microtubules with various proteins.     

Choosing and using Schizosaccharomyces pombe plasmids

A wide range of plasmids has been developed for molecular studies in the fission yeast Schizosaccharomyces pombe. This includes general purpose episomes, expression vectors, epitope tagging plasmids, and integration vectors. This review describes the typical features of S. pombe vectors, including replication origins, positive and negative selection markers, and constitutive and inducible promoter systems. We will also discuss vectors with epitope tags and how these can be used to modify episomal or endogenous gene sequences. Considerations for choosing and using a plasmid are presented and specialized methods are described.     

Conjugation-induced lysis of Schizosaccharomyces pombe.

About 15% of the conjugating cells of Schizosaccharomyces pombe were observed to lyse spontaneously during the conjugation process. Lysis occurred at the site of union.     

Malate transport in Schizosaccharomyces pombe.

The transport of malate was studied in a Schizosaccharomyces pombe wild-type strain and in mutant strains unable to utilize malic acid. Two groups of such mutants, i.e., malic enzyme-deficient and malate transport-defective mutants, were differentiated by a 14C-labeled L-malate transport assay and by starch gel electrophoresis followed by activity staining for malic enzyme (malate dehydrogenase [oxaloacetate decarboxylating] [NAD+]; 1.1.1.38) and malate dehydrogenase (1.1.1.37). Transport of malate in S. pombe was constitutive and strongly inhibited by inhibitors of oxidative phosphorylation and of the formulation of proton gradients. Transport was a saturable function of the malate concentration. The apparent Km and Vmax values for transport by the parent were 3.7 mM and 40 nmol/min per mg of protein, respectively, while those of the malic enzyme-deficient mutant were 5.7 mM and 33 nmol/min per mg of protein, respectively. Malate transport was pH and temperature dependent. The specificity of transport was studied with various substrates, including mono- and dicarboxylic acids, and the possibility of a common transport system for dicarboxylic acids is discussed.     

Protein prenylation in Schizosaccharomyces pombe.

S. pombe is shown to be a powerful system for studies concerning attachment of polyisoprenoid moieties to proteins, due to its ability to take up exogenous mevalonic acid efficiently. The fission yeast can take up about 5% of the exogenously added mevalonic acid and incorporate approximately 10% of this into protein. By contrast, the uptake obtained with the budding yeast S. cerevisiae is less than 0.5%. HPLC analysis of total S. pombe protein-bound isoprenoids revealed that approximately 55% of the counts co-migrated with the geranylgeraniol standard, while approximately 45% of the counts co-migrated with farnesol. We could not detect any effects of mevinolin or other HMG-CoA reductase inhibitors in S. pombe.     

Pre-mRNA splicing mutants of Schizosaccharomyces pombe.

A collection of temperature sensitive (ts-) mutants was prepared by chemical mutagenesis of a wild type Schizosaccharomyces pombe strain. To screen the ts- mutants for pre-mRNA splicing defects, an oligodeoxynucleotide that recognizes one of the introns of the beta-tubulin pre-mRNA was used as a probe in a Northern blot assay to detect accumulation of intron sequences. This screening procedure identified three pre-mRNA splicing mutants from 100 ts- strains. The three mutants are defective in an early step of the pre-mRNA splicing reaction; none accumulate intermediates. The precursors that accumulate at 37 degrees C are polyadenylated. Analysis of the splicing of another pre-mRNA showed that the mutations are not specific for beta-tubulin. The total RNA pattern in the three splicing mutants appears to be normal. In addition, the amounts of the spliceosomal snRNAs are not drastically changed compared to the wild type and splicing of pre-tRNAs is not blocked. Genetic analyses demonstrate that all three splicing mutations are tightly linked to the ts- growth defects and are recessive. Crosses among the mutants place them in three complementation groups. The mutants have been named prp1, prp2 and prp3.     

Replication of centromere II of Schizosaccharomyces pombe.

The centromeric DNAs of Schizosaccharomyces pombe chromosomes resemble those of higher eukaryotes in being large and composed predominantly of repeated sequences. To begin a detailed analysis of the mode of replication of a complex centromere, we examined whether any sequences within S. pombe centromere II (cen2) have the ability to mediate autonomous replication. We found a high density of segments with such activity, including at least eight different regions comprising most of the repeated and unique centromeric DNA elements. A physical mapping analysis using two-dimensional gels showed that autonomous replication initiated within the S. pombe sequences in each plasmid. A two-dimensional gel analysis of replication on the chromosomes revealed that the K and L repeat elements, which occur in multiple copies at all three centromeres and comprise approximately 70% of total centromeric DNA mass in S. pombe, are both sites of replication initiation. In contrast, the unique cen2 central core, which contains multiple segments that can support autonomous replication, appears to be repressed for initiation on the chromosome. We discuss the implications of these findings for our understanding of DNA replication and centromere function.     

Post-translational processing of Schizosaccharomyces pombe YPT proteins.

ras proteins are post-translationally processed at their carboxyl-terminal CAAX motif by a triplet of modifications: prenylation of C with farnesyl, proteolytic trimming of AAX, and carboxyl-methylation. These modifications co-operate with palmitoylation of nearby sites or a polybasic region to target plasma membrane localization. The related YPT/rab proteins in contrast are localized to compartments of the endo-membrane system and may be involved in directing membrane traffic. These proteins end in XCC or CXC motifs. We have analyzed the processing of members of this subfamily form the fission yeast Schizosaccharomyces pombe. We find using in vitro translation in reticulocyte lysates that YPT1, -3, and -5 are prenylated with geranylgeranyl and that they incorporate label from [3H]mevalonic acid when expressed in transfected COS cells in vivo. Furthermore, prenylation was necessary for membrane binding in vivo. The CXC protein YPT5, but neither of the two XCC proteins YPT1 and YPT3, was carboxyl-methylated in S. pombe and in COS cells in vivo. However, YPT5 was not carboxyl-methylated in vitro in lysates which were able to methylate ras protein. YPT3 was detectably palmitoylated when expressed in COS cells, though at a much lower level than ras.     

Dielectric properties of yeast cells as determined by electrorotation.

Electrorotational spectra of yeast cells, Saccharomyces cerevisiae strain R XII, were measured over a frequency range of nearly 7 decades. The physical properties of distinct cell parts were simultaneously determined for individual cells by comparison with an electrical two-shell model: The conductivity of the cytoplasm, cell wall and cytoplasmic membrane of living cells were found to be 5.5 mS/cm, 0.1 to more than 0.5 mS/cm and less than 0.25 nS/cm to 4.5 microS/cm, respectively. The conductivity of the cytoplasmic membrane was dependent on the conductivity of the medium. Membrane behaviour is interpreted as an opening of membrane channels when the environment becomes more physiological. The specific membrane capacitance was determined to be 1.1 microF/cm2 and the thickness of the cell wall was calculated as 0.11 micron. Heat treated cells showed an increased membrane conductivity of more than 0.1 microS/cm (at 25 microS/cm medium conductivity) and a drop in cytoplasmic conductivity to between 0.1 and 0.8 mS/cm, depending on the length of time the cells were suspended in low conductivity water (25 microS/cm), indicating a perforation of the membrane. A slightly decreased spinning speed scaling factor for dead cells suggests a modification to the cellular surface, while the principal structure of the cell wall appears to be uneffected. It can be demonstrated by these observations, that cellular electrorotation permits the simultaneous investigation of the different cellular compartments of individual cells in vivo under various environmental conditions.     

Buoyant density constancy of Schizosaccharomyces pombe cells.

Buoyant densities of cells from exponentially growing cultures of the fission yeast Schizosaccharomyces pombe 972h- with division rates from 0.14 to 0.5 per h were determined by equilibrium centrifugation in Percoll gradients. Buoyant densities were independent of growth rate, with an average value (+/- standard error) of 1.0945 (+/- 0.00037) g/ml. When cells from these cultures were separated by size, mean cell volumes were independent of buoyant density, indicating that buoyant densities also were independent of cell age during the division cycle. These results support the suggestion that most or all kinds of cells that divide by equatorial fission may have similar, evolutionarily conserved mechanisms for regulation of buoyant density.