Fission Yeast Mutants Research Articles

Correction: Polypeptone Induces Dramatic Cell Lysis in ura4 Deletion Mutants of Fission Yeast

A funding organization for the fifth author was incorrectly omitted from the Funding Statement. The following sentence should be read after the first sentence of the Funding Statement: TT is supported by Cancer Research UK.

The effect of coenzyme Q10 included by γ-cyclodextrin on the growth of fission yeast studied by microscope Raman spectroscopy

The inclusion complex of coenzyme Q10 (CoQ10) by γ-cyclodextrin (γ-CD), CoQ10-CD complex, was recently developed. The addition of the CoQ10-CD complex recovered the growth of a fission yeast mutant strain, Δdps1, which otherwise cannot grow well due to the lack of coenzyme Q producing ability. However, the oxygen consumption rate of this strain was not restored by the addition of the CoQ10-CD complex. The addition of two other anti-oxidative reagents, glutathione and ascorbic acid, also recovered the growth of the Δdps1 strain as well. These results indicated that the recovery of the growth of Δdps1 was brought about by the anti-oxidative property of CoQ10. The intensity of Raman spectra of Δdps1 at 1602cm−1, which is prominently observed for the wild type of the fission yeast, was compared between before and after addition of the CoQ10-CD complex. The signal was very weakly observed for Δdps1 and did not increase in intensity by the addition of the CoQ10-CD complex. These results suggested the recovery of the growth of Δdps1 was brought about not by the restoration of respiration function of Δdps1 but by the anti-oxidative property of CoQ10 to result in the decrease in the oxidative stress.

Insight into Actin Organization and Function in Cytokinesis from Analysis of Fission Yeast Mutants

Actin is a key cytoskeletal protein with multiple roles in cellular processes such as polarized growth, cytokinesis, endocytosis, and cell migration. Actin is present in all eukaryotes as highly dynamic filamentous structures, such as linear cables and branched filaments. Detailed investigation of the molecular role of actin in various processes has been hampered due to the multifunctionality of the protein and the lack of alleles defective in specific processes. The actin cytoskeleton of the fission yeast, Schizosaccharomyces pombe, has been extensively characterized and contains structures analogous to those in other cell types. In this study, primarily with the view to uncover actin function in cytokinesis, we generated a large bank of fission yeast actin mutants that affect the organization of distinct actin structures and/or discrete physiological functions of actin. Our screen identified 17 mutants with specific defects in cytokinesis. Some of these cytokinesis mutants helped in dissecting the function of specific actin structures during ring assembly. Further genetic analysis of some of these actin mutants revealed multiple genetic interactions with mutants previously known to affect the actomyosin ring assembly. We also characterize a mutant allele of actin that is suppressed upon overexpression of Cdc8p-tropomyosin, underscoring the utility of this mutant bank. Another 22 mutant alleles, defective in polarized growth and/or other functions of actin obtained from this screen, are also described in this article. This mutant bank should be a valuable resource to study the physiological and biochemical functions of actin.

Polypeptone Induces Dramatic Cell Lysis in ura4 Deletion Mutants of Fission Yeast

Polypeptone is widely excluded from Schizosaccharomyces pombe growth medium. However, the reasons why polypeptone should be avoided have not been documented. Polypeptone dramatically induced cell lysis in the ura4 deletion mutant when cells approached the stationary growth phase, and this phenotype was suppressed by supplementation of uracil. To determine the specificity of this cell lysis phenotype, we created deletion mutants of other genes involved in de novo biosynthesis of uridine monophosphate (ura1, ura2, ura3, and ura5). Cell lysis was not observed in these gene deletion mutants. In addition, concomitant disruption of ura1, ura2, ura3, or ura5 in the ura4 deletion mutant suppressed cell lysis, indicating that cell lysis induced by polypeptone is specific to the ura4 deletion mutant. Furthermore, cell lysis was also suppressed when the gene involved in coenzyme Q biosynthesis was deleted. This is likely because Ura3 requires coenzyme Q for its activity. The ura4 deletion mutant was sensitive to zymolyase, which mainly degrades (1,3)-beta-D glucan, when grown in the presence of polypeptone, and cell lysis was suppressed by the osmotic stabiliser, sorbitol. Finally, the induction of cell lysis in the ura4 deletion mutant was due to the accumulation of orotidine-5-monophosphate. Cell wall integrity was dramatically impaired in the ura4 deletion mutant when grown in the presence of polypeptone. Because ura4 is widely used as a selection marker in S. pombe, caution needs to be taken when evaluating phenotypes of ura4 mutants.

Haploinsufficiency of the Sec7 guanine nucleotide exchange factor gea1 impairs septation in fission yeast.

Membrane trafficking is essential to eukaryotic life and is controlled by a complex network of proteins that regulate movement of proteins and lipids between organelles. The GBF1/GEA family of Guanine nucleotide Exchange Factors (GEFs) regulates trafficking between the endoplasmic reticulum and Golgi by catalyzing the exchange of GDP for GTP on ADP Ribosylation Factors (Arfs). Activated Arfs recruit coat protein complex 1 (COP-I) to form vesicles that ferry cargo between these organelles. To further explore the function of the GBF1/GEA family, we have characterized a fission yeast mutant lacking one copy of the essential gene gea1 (gea1+/−), the Schizosaccharomyces pombe ortholog of GBF1. The haploinsufficient gea1+/− strain was shown to be sensitive to the GBF1 inhibitor brefeldin A (BFA) and was rescued from BFA sensitivity by gea1p overexpression. No overt defects in localization of arf1p or arf6p were observed in gea1+/− cells, but the fission yeast homolog of the COP-I cargo sac1 was mislocalized, consistent with impaired COP-I trafficking. Although Golgi morphology appeared normal, a slight increase in vacuolar size was observed in the gea1+/− mutant strain. Importantly, gea1+/− cells exhibited dramatic cytokinesis-related defects, including disorganized contractile rings, an increased septation index, and alterations in septum morphology. Septation defects appear to result from altered secretion of enzymes required for septum dynamics, as decreased secretion of eng1p, a β-glucanase required for septum breakdown, was observed in gea1+/− cells, and overexpression of eng1p suppressed the increased septation phenotype. These observations implicate gea1 in regulation of septum breakdown and establish S. pombe as a model system to explore GBF1/GEA function in cytokinesis.

Mapping genomic hotspots of DNA damage by a single-strand-DNA-compatible and strand-specific ChIP-seq method

Spontaneous DNA damage may occur nonrandomly in the genome, especially when genome maintenance mechanisms are undermined. We developed single-strand DNA (ssDNA)–associated protein immunoprecipitation followed by sequencing (SPI-seq) to map genomic hotspots of DNA damage. We demonstrated this method with Rad52, a homologous recombination repair protein, which binds to ssDNA formed at DNA lesions. SPI-seq faithfully detected, in fission yeast, Rad52 enrichment at artificially induced double-strand breaks (DSBs) as well as endogenously programmed DSBs for mating-type switching. Applying Rad52 SPI-seq to fission yeast mutants defective in DNA helicase Pfh1 or histone H3K56 deacetylase Hst4, led to global views of DNA lesion hotspots emerging in these mutants. We also found serendipitously that histone dosage aberration can activate retrotransposon Tf2 and cause the accumulation of a Tf2 cDNA species bound by Rad52. SPI-seq should be widely applicable for mapping sites of DNA damage and uncovering the causes of genome instability.

Determining proteome-wide expression levels using reverse protein arrays in fission yeast

Global protein expression profiling of various mutants or growth conditions is currently a major challenge in biology. Here we provide a protocol for a strategy that we recently developed that couples ORFeome-based (ORF denotes open reading frame) expression to reverse protein arrays; this approach accurately quantifies more than 99% of the predicted fission yeast proteins in various genetic backgrounds. The first stage of this two-stage protocol requires mass mating between any fertile fission yeast mutant of interest and the integrated fission yeast-tagged ORFeome followed by selection of recombinant haploids. The second stage of the protocol, called reverse protein arrays, involves simple large-scale extraction of total proteins, which are then spotted on nitrocellulose membranes for detection by quantitative dot blot. When handled manually, the entire protocol takes about 2 months. However, the process could easily be automated and should also be applicable to other organisms.

Response to Arsenate Treatment in Schizosaccharomyces pombe and the Role of Its Arsenate Reductase Activity

Arsenic toxicity has been studied for a long time due to its effects in humans. Although epidemiological studies have demonstrated multiple effects in human physiology, there are many open questions about the cellular targets and the mechanisms of response to arsenic. Using the fission yeast Schizosaccharomyces pombe as model system, we have been able to demonstrate a strong activation of the MAPK Spc1/Sty1 in response to arsenate. This activation is dependent on Wis1 activation and Pyp2 phosphatase inactivation. Using arsenic speciation analysis we have also demonstrated the previously unknown capacity of S. pombe cells to reduce As (V) to As (III). Genetic analysis of several fission yeast mutants point towards the cell cycle phosphatase Cdc25 as a possible candidate to carry out this arsenate reductase activity. We propose that arsenate reduction and intracellular accumulation of arsenite are the key mechanisms of arsenate tolerance in fission yeast.

GENERAL LAW OF GROWTH AND REPLICATION, GROWTH EQUATION AND ITS APPLICATIONS

We present significantly advanced studies of the previously introduced physical growth mechanism and unite it with biochemical growth factors. Obtained results allowed formulation of the general growth law which governs growth and evolutional development of all living organisms, their organs and systems. It was discovered that the growth cycle is predefined by the distribution of nutritional resources between maintenance needs and biomass production. This distribution is quantitatively defined by the growth ratio parameter, which depends on the geometry of an organism, phase of growth and, indirectly, the organism's biochemical machinery. The amount of produced biomass, in turn, defines the composition of biochemical reactions. Changing amount of nutrients diverted to biomass production is what forces organisms to proceed through the whole growth and replication cycle. The growth law can be formulated as follows: the rate of growth is proportional to influx of nutrients and growth ratio. Considering specific biochemical components of different organisms, we find influxes of required nutrients and substitute them into the growth equation; then, we compute growth curves for amoeba, wild type fission yeast, and fission yeast's mutant. In all cases, predicted growth curves correspond very well to experimental data. Obtained results prove validity and fundamental scientific value of the discovery.

Treslin, DUE-B, and GEMC1 cannot complement Sld3 mutants in fission yeast

Initiation of DNA replication in eukaryotes is an evolutionarily conserved process that involves two distinct steps: the formation of prereplication complexes at replication origins in G1 and the assembly of preinitiation complexes (pre-ICs) in S phase, which leads to activation of the replication helicase. For the assembly of pre-ICs in yeast, formation of the Sld2-Dpb11-Sld3 complex is a critical event that requires phosphorylation of Sld2 and Sld3 by cyclin-dependent kinase. In mammals, RecQL4 and TopBP1 are excellent ortholog candidates for Sld2 and Dpb11, respectively. In this past year, three TopBP1-interacting proteins Treslin/Ticrr, GEMC1, and DUE-B have been identified in metazoans as possible functional orthologs of the yeast Sld3. To test this hypothesis, we carried out several complementation tests in fission yeast. The proteins were expressed at various levels in the temperature-sensitive sld3-10 mutant and in cells that lack endogenous Sld3. Our result showed that none of these metazoan proteins could rescue growth defect of the sld3 mutants. Although the result may have several interpretations, it is possible that the helicase activation in mammals has diverged in complexity during evolution from that in yeasts and may involve multiple players that interact with TopBP1.

The Conserved Function of Skp1 in Meiosis

Many components of regulatory networks governing basic cellular functions are highly homologous in diverse species. An important question is how such conservation of individual components is translated into the conservation at the level of regulatory networks. An intriguing case regarding this question is concerned with the function of the Skp1 protein in meiosis. Skp1 is a conserved subunit of the Skp1-Cullin-F-box-protein (SCF) E3 ubiquitin ligase (Tyers and Jorgensen, 2000; Willems et al., 2004). For nearly 13 years, a distinct meiotic phenotype was described only in a null mutant of Skp1 in Arabidopsis thaliana, namely arabidopsis-skp1-like1-1 (ask1-1) (Yang et al., 1999; Yang and Ma, 2001; Wang et al., 2004; Wang and Yang, 2006). It raises the question of whether such a phenotype indicates a plant-specific function of Skp1 (or the corresponding SCF) in meiosis. However, a strikingly similar meiotic phenotype was recently reported by Okamoto et al. (2012) in skp1 mutants of fission yeast (Schizosaccharomyces pombe). These phenotypic similarities suggest the presence of a conserved network involving an SCF that regulates the progression of recombination in meiosis. The first similarity is that the numbers of spores produced by a meiocyte range from two to more than four spores in ask1-1 (Yang et al., 1999) and the yeast skp1 mutants. In many meiotic mutants, the meiotic products produced by a meiocyte are either more or fewer than the normal four spores. The wide range of meiotic product number per meiocyte places ask1-1 and the yeast skp1 mutants in a rare group. The second similarity is indicated by the morphology and behavior of chromosomes during meiosis I. Chromosomes entangle, non-disjoin, are stretched by the anaphase spindle, and as a result, are unevenly distributed to meiotic products in both ask1-1 (Yang et al., 1999; Wang et al., 2004) and the yeast skp1 mutants. Chromosomes are asynaptic in prophase I in ask1-1 (Wang et al., 2004). Fission yeast normally undergoes homologous chromosome pairing instead of synapsis in prophase I. It is unknown whether the homologous chromosome pairing in the yeast skp1 mutants is also affected, but it may be so based on the overall similarities in chromosome behavior between ask1-1 and the yeast skp1 mutants. Spindle behavior is intimately linked to chromosome behavior. It is unsurprising that the spindle seems similarly affected by chromosome entanglement in ask1-1 and the yeast skp1 mutants. Although the experimental approaches somewhat differ in the investigations of the two organisms – study of still images in Arabidopsis and study of time-lapsed videos in yeast, similar conclusions can be made on the impact of entangled chromosomes on the spindle morphology. The Arabidopsis work found anaphase spindles that were either shorter than metaphase spindles or longer than typical anaphase spindles in ask1-1 (Yang and Ma, 2001). The yeast work revealed bending, collapsing, reforming, and re-elongation of spindles. It can be said that spindles are deformed in a similar fashion when they struggle to separate entangled chromosomes in the skp1 mutants of both organisms. Lastly, the underlying cause of the chromosome entanglement may be attributed to the presence of unresolved recombination intermediates in ask1-1 and the yeast skp1 mutants. In ask1-1, electron-dense materials abnormally accumulate on chromosomes in prophase I (Wang et al., 2004), the dynamics of a cohesin on meiotic chromosomes is altered (Zhao et al., 2006), and recombination is promoted in plants heterozygous for ask1-1 (Wang and Yang, 2006). In comparison, several proteins involved in recombination are retained longer than usual on meiotic chromosomes in the yeast skp1 mutants. Thus, evidence from both Arabidopsis and yeast suggests that Skp1 is involved in promoting the resolution of recombination intermediates in meiosis. Perhaps a moderate reduction in the ASK1 function in Arabidopsis plants heterozygous for ask1-1 enhances the formation of recombination intermediates not to a toxic level so that recombination frequency is increased. The Skp1 protein is not expected to be directly involved in the recombination process, and yet the phenotypes of its Arabidopsis and yeast mutants closely resemble each other. It is also noted that the yeast work was carried out without the knowledge of the Arabidopsis work (The Arabidopsis work was not cited by the yeast paper). The converging evidence from both Arabidopsis and yeast suggests the existence of a conserved network in the regulation of recombination progression in meiosis. An F-box protein with a DNA helicase domain is suggested by Okamoto et al. (2012) to be the one linking Skp1 to the recombination process in yeast. The Arabidopsis genome does not contain a homolog of the yeast F-box-associated DNA helicase (based on BLAST search using the yeast protein sequence as query) although it has many proteins containing either an F-box or a DNA helicase domain. Further elucidation of the predicted network in diverse species will likely reveal novel aspects in the regulation of meiotic recombination.

Isolation of synthetic lethal mutations in the rsm1-null mutant of fission yeast

To identify mutations in genes that are genetically linked to rsm1, we performed a synthetic lethal genetic screen in the fission yeast, Schizosaccharomyces pombe. Four mutations that showed synthetic lethality in combination with the rsm1null allele were isolated from approximately 320,000 colonies and defined in three complementation groups. One mutant (SLrsm1) exhibited a significant accumulation of poly(A)(+) RNA in the nucleus under synthetic lethal conditions, while the rest had no mRNA export defects. In addition, some genes (spmex67, rae1, or mlo3) required for mRNA export complemented the growth defects of the identified mutants. These results suggest that the isolated mutants contain mutations in genes that are involved in mRNA export and/or pre-mRNA retention.

Laser microsurgery provides evidence for merotelic kinetochore attachments in fission yeast cells lacking Pcs1 or Clr4

In order to segregate chromosomes properly, the cell must prevent merotelic kinetochore attachment, an error that occurs when a single kinetochore is attached to microtubules emanating from both spindle poles. Merotelic kinetochore orientation represents a major mechanism of aneuploidy in mitotic mammalian cells and it is the primary mechanism of chromosome instability in cancer cells. Fission yeast mutants defective in putative microtubule-site clamp Pcs1/Mde4 or Clr4/Swi6-dependent centromeric heterochromatin display high frequencies of lagging chromosomes during anaphase. Here, we developed an assay based on laser microsurgery to show that the stretched morphology of lagging kinetochores in pcs1Δ and clr4Δ mutant cells is due to merotelic attachment. We further show that Mde4 is regulated by Cdc2 and that Cdc2 activity prevents precocious localization of Mde4 to the metaphase spindle. Finally, we show that Pcs1/Mde4 complex shares similar features with the conserved kinetochore complex Spc24/Spc25 suggesting that these two complexes may occupy a similar functional niche.

Isolation of a fission yeast mutant that is sensitive to valproic acid and defective in the gene encoding Ric1, a putative component of Ypt/Rab-specific GEF for Ryh1 GTPase

Valproic acid (VPA) causes various therapeutic and biological effects, but the exact mechanisms underlying these effects, however, remain elusive. To gain insights into the molecular mechanisms of VPA action, we performed in fission yeast a genetic screen for mutants that show VPA hypersensitivity and have identified several membrane-trafficking mutants including vas1-1/vps45 and vas2-1/aps1. Here, we describe the isolation and characterization of vas3-1/ric1-v3, a mutant allele of the ric1 (+) gene encoding a fission yeast homolog of the budding yeast Ric1p, a component of Ypt/Rab-specific guanyl-nucleotide exchange factor (GEF). The Rab GTPase Ryh1 knockout (Deltaryh1) cells and Deltaric1 cells exhibited similar phenotypes. The double knockout Deltaric1Deltaryh1 cells did not display synthetic growth defects. These results are consistent with the notion that Ric1 may be a component of the GEF complex for Ryh1. Overexpression of wild-type Ryh1 and the constitutively active Ryh1Q70L only partially suppressed the phenotypes of ric1-v3 and Deltaric1 cells, and they failed to localize to the Golgi/endosomes in ric1-v3 and Deltaric1 cells. Furthermore, we isolated vps15 (+) gene, encoding a serine/threonine protein kinase, as a dosage-dependent suppressor of the temperature-sensitive phenotype of ric1-v3 mutant, but not that of Deltaric1 cells. Our results showed that the ric1-v3 mutant allele has some residual functional activity and suggest that Vps15 plays a role in the regulation of Ric1 function. In conclusion, Ric1 is a putative component of GEF for Ryh1 and might be regulated by Vps15. Further studies are needed to reveal the mechanism underlying the regulation.

Fission yeast Vps1 and Atg8 contribute to oxidative stress resistance

Organisms have evolved diverse means to protect themselves from oxidative stress. To better understand the molecular mechanisms involved in oxidative stress resistance, we screened fission yeast mutants sensitive to paraquat, a reagent acting on the mitochondria to generate reactive oxygen species. Among the mutants we isolated, we focused on a mutant defective in the vps1(+) (vacuolar protein sorting 1) gene that encodes a dynamin-related protein family member. vps1Δ exhibited aberrant mitochondrial and vacuolar morphology on treatment with paraquat. vps1Δ was sensitive to osmotic stress, high concentrations of Ca(2+) and Fe(2+). Interestingly, the deletion of atg8(+), a gene essential for the autophagy pathway, exhibited strong genetic interactions with vps1Δ. The vps1Δatg8Δ double mutant was additively sensitive to oxidative stress, osmotic stress and Ca(2+). The deletion of vps1(+) rescued the bizarre vacuolar morphology shown by atg8Δ. Such genetic interactions were not observed with other atg mutants. Furthermore, the atg8-G116A mutant did not show abnormal vacuolar morphology while being sensitive to nitrogen starvation, an autophagy-related phenotype. Taken together, we conclude that atg8(+) regulates vacuolar functions independently of its role in autophagy. We propose that Vps1 and Atg8 cooperatively participate in vacuolar function, thereby contributing to oxidative stress resistance.

Spatial Control of Cytokinesis by Cdr2 Kinase and Mid1/Anillin Nuclear Export

Maintaining genome integrity and cellular function requires proper positioning of the cell division plane. In most eukaryotes, cytokinesis relies on a contractile actomyosin ring positioned by intrinsic spatial signals that are poorly defined at the molecular level. Fission yeast cells assemble a medial contractile ring in response to positive spatial cues from the nucleus at the cell center and negative spatial cues from the cell tips. These signals control the localization of the anillin-like protein Mid1, which defines the position of the division plane at the medial cortex, where it recruits contractile-ring components at mitosis onset. Here we show that Cdr2 kinase anchors Mid1 at the medial cortex during interphase through association with the Mid1 N terminus. This association underlies the negative regulation of Mid1 distribution by cell tips. We also demonstrate that the positive signaling from the nucleus is based on Mid1 nuclear export, which links division-plane position to nuclear position during early mitosis. After nuclear displacement, Mid1 nuclear export is dominant over Cdr2-dependent positioning of Mid1. We conclude that Cdr2- and nuclear export-dependent positioning of Mid1 constitute two overlapping mechanisms that relay cell polarity and nuclear positional information to ensure proper division-plane specification.

Comparison of a coq7 deletion mutant with other respiration‐defective mutants in fission yeast

Among the steps in ubiquinone biosynthesis, that catalyzed by the product of the clk-1/coq7 gene has received considerable attention because of its relevance to life span in Caenorhabditis elegans. We analyzed the coq7 ortholog (denoted coq7) in Schizosaccharomyces pombe, to determine whether coq7 has specific roles that differ from those of other coq genes. We first confirmed that coq7 is necessary for the penultimate step in ubiquinone biosynthesis, from the observation that the deletion mutant accumulated the ubiquinone precursor demethoxyubiquinone-10 instead of ubiquinone-10. The coq7 mutant displayed phenotypes characteristic of other ubiquinone-deficient Sc. pombe mutants, namely, hypersensitivity to hydrogen peroxide, a requirement for antioxidants for growth on minimal medium, and an elevated production of sulfide. To compare these phenotypes with those of other respiration-deficient mutants, we constructed cytochrome c (cyc1) and coq3 deletion mutants. We also assessed accumulation of oxidative stress in various ubiquinone-deficient strains and in the cyc1 mutant by measuring mRNA levels of stress-inducible genes and the phosphorylation level of the Spc1 MAP kinase. Induction of ctt1, encoding catalase, and apt1, encoding a 25 kDa protein, but not that of gpx1, encoding glutathione peroxidase, was indistinguishable in four ubiquinone-deficient mutants, indicating that the oxidative stress response operates at similar levels in the tested strains. One new phenotype was observed, namely, loss of viability in stationary phase (chronological life span) in both the ubiquinone-deficient mutant and in the cyc1 mutant. Finally, Coq7 was found to localize in mitochondria, consistent with the possibility that ubiquinone biosynthesis occurs in mitochondria in yeasts. In summary, our results indicate that coq7 is required for ubiquinone biosynthesis and the coq7 mutant is not distinguishable from other ubiquinone-deficient mutants, except that its phenotypes are more pronounced than those of the cyc1 mutant.

Phosphorylation of HsMis13 by Aurora B Kinase Is Essential for Assembly of Functional Kinetochore

Chromosome movements in mitosis are orchestrated by dynamic interactions between spindle microtubules and the kinetochore, a multiprotein complex assembled onto centromeric DNA of the chromosome. Here we show that phosphorylation of human HsMis13 by Aurora B kinase is required for functional kinetochore assembly in HeLa cells. Aurora B interacts with HsMis13 in vitro and in vivo. HsMis13 is a cognate substrate of Aurora B, and the phosphorylation sites were mapped to Ser-100 and Ser-109. Suppression of Aurora B kinase by either small interfering RNA or chemical inhibitors abrogates the localization of HsMis13 but not HsMis12 to the kinetochore. In addition, non-phosphorylatable but not wild type and phospho-mimicking HsMis13 failed to localize to the kinetochore, demonstrating the requirement of phosphorylation by Aurora B for the assembly of HsMis13 to kinetochore. In fact, localization of HsMis13 to the kinetochore is spatiotemporally regulated by Aurora B kinase, which is essential for recruiting outer kinetochore components such as Ndc80 components and CENP-E for functional kinetochore assembly. Importantly, phospho-mimicking mutant HsMis13 restores the assembly of CENP-E to the kinetochore, and tension developed across the sister kinetochores in Aurora B-inhibited cells. Thus, we reason that HsMis13 phosphorylation by Aurora B is required for organizing a stable bi-oriented microtubule kinetochore attachment that is essential for faithful chromosome segregation in mitosis.

Plasmodium falciparum dolichol phosphate mannose synthase represents a novel clade

Dolichol phosphate mannose synthase (DPM) catalyzes the reaction between dolichol phosphate (Dol-P) and guanosine diphosphate mannose (GDP-Man) to form dolichol-phosphate-mannose (Dol-P-Man). This molecule acts as mannose donor for N-glycosylation and glycosylphosphatidylinositol (GPI) biosynthesis. The Plasmodium falciparum DPM1 (Pfdpm1) possesses a single predicted transmembrane region near the N-, but not the C-terminus. Here we show that the cloned Pfdpm1 gene failed to complement a Saccharomyces cerevisiae mutant indicating that the parasite gene does not belong to the baker’s yeast group, as was previously assumed. Furthermore, Pfdpm1 was unable to complement a mouse mutant deficient in DPM but efficiently complements the Schizosaccharomyces pombe fission yeast mutant, indicating a difference between fission yeast and mammalian DPM genes. Therefore, we reanalyzed the hydrophobicity scales of all known DPMs and consequently reclassify the DPM clade into six major novel subgroups. Furthermore, we show that Pfdpm1 represents a unique enzyme among these subgroups.

Expression of a Novel 90-kDa Protein, Lsd90, Involved in the Metabolism of Very Long-chain Fatty Acid-containing Phospholipids in a Mitosis-defective Fission Yeast Mutant

The fission yeast lsd1/fas2 strain carries a temperature-sensitive mutation of the fatty-acid-synthase alpha-subunit, exhibiting an aberrant mitosis lsd phenotype, with accumulation of very-long-chain fatty-acid-containing phospholipid (VLCFA-PL). A novel 90-kDa protein, Lsd90 (SPBC16E9.16c), was found to be newly expressed in small particle-like structures in lsd1/fas2 cells under restrictive conditions. Two mismatches leading to a double frame shift were found between the sequences of the lsd90(+) gene registered in the genomic database and the sequences determined experimentally at the amino acid, cDNA and genomic DNA levels. Unexpectedly, overexpression and disruption of the lsd90(+) gene in either lsd1/fas2 or wild-type cells did not affect either cell growth or expression of the lsd phenotype. The amounts of VLCFA-PL that accumulated in lsd90-overexpressing lsd1/fas2 cells were significantly lower than those in lsd1/fas2 cells, suggesting the involvement of Lsd90 in the metabolism of VLCFA-PL.