Schizosaccharomyces pombe Pmr1p is essential for cell wall integrity and is required for polarized cell growth and cytokinesis

The cps5-138 fission yeast mutant shows an abnormal lemon-like morphology at 28 degrees C in minimal medium and a lethal thermosensitive phenotype at 37 degrees C. Cell growth is completely inhibited at 28 degrees C in a Ca2+-free medium, in which the wild type is capable of growing normally. Under these conditions, actin patches become randomly distributed throughout the cell, and defects in septum formation and subsequent cytokinesis appear. The mutant cell is hypersensitive to the cell wall-digesting enzymatic complex Novozym234 even under permissive conditions. The gene SPBC31E1.02c, which complements all the mutant phenotypes described above, was cloned and codes for the Ca2+-ATPase homologue Pmr1p. The gene is not essential under optimal growth conditions but is required under conditions of low Ca2+ (<0.1 mM) or high temperature (>35 degrees C). The green fluorescent protein-tagged Cps5 proteins, which are expressed under physiological conditions (an integrated single copy with its own promoter in the cps5Delta strain), display a localization pattern typical of endoplasmic reticulum proteins. Biochemical analyses show that 1,3-beta-D-glucan synthase activity in the mutant is decreased to nearly half that of the wild type and that the mutant cell wall contains no detectable galactomannan when the cells are exposed to a Ca2+-free medium. The mutant acid phosphatase has an increased electrophoretic mobility, suggesting that incomplete protein glycosylation takes place in the mutant cells. These results indicate that S. pombe Pmr1p is essential for the maintenance of cell wall integrity and cytokinesis, possibly by allowing protein glycosylation and the polarized actin distribution to take place normally. Disruption and complementation analyses suggest that Pmr1p shares its function with a vacuolar Ca2+-ATPase homologue, Pmc1p (SPAPB2B4.04c), to prevent lethal activation of calcineurin for cell growth.     

Swm1p, a subunit of the APC/cyclosome, is required to maintain cell wall integrity during growth at high temperature in Saccharomyces cerevisiae

Swm1p, a subunit of the APC cyclosome, was originally identified for its role in the later stages of the sporulation process and is required for spore wall assembly. In addition, this protein is required to maintain cell wall integrity in vegetative cells during growth at high temperature. Electron microscopy analyses of mutant cells grown at the restrictive temperature in the absence of osmotic support show that the cell wall is clearly abnormal, with large number of discontinuities that may be responsible for the observed lysis. The mutant cells show a 7-fold reduction in glucan synthase activity during growth at 38 degrees C and a 3.5-fold increase in the chitin content of the cell wall. The chitin is deposited in a delocalized manner all over the cell wall, where it accumulates in patches in abnormal regions. The excess chitin is mainly synthesized by the action of chitin synthase III (Chs3p), since it disappears in the swm1 chs3 double-mutant.     

Yeast adaptor protein, Nbp2p, is conserved regulator of fungal Ptc1p phosphatases and is involved in multiple signaling pathways

Nbp2p is an Src homology 3 (SH3) domain-containing yeast protein that is involved in a variety of cellular processes. This small adaptor protein binds to a number of different proteins through its SH3 domain, and a region N-terminal to the SH3 domain binds to the protein phosphatase, Ptc1p. Despite its involvement in a large number of physical and genetic interactions, the only well characterized function of Nbp2p is to recruit Ptc1p to the high osmolarity glycerol pathway, which results in down-regulation of this pathway. In this study, we have discovered that Nbp2p orthologues exist in all Ascomycete and Basidiomycete fungal genomes and that all possess an SH3 domain and a conserved novel Ptc1p binding motif. The ubiquitous occurrence of these two features, which we have shown are both critical for Nbp2p function in Saccharomyces cerevisiae, implies that a conserved role of Nbp2p in all of these fungal species is the targeting of Ptc1p to proteins recognized by the SH3 domain. We also show that in a manner analogous to its role in the high osmolarity glycerol pathway, Nbp2p functions in the down-regulation of the cell wall integrity pathway through SH3 domain-mediated interaction with Bck1p, a component kinase of this pathway. Based on functional studies on the Schizosaccharomyces pombe and Neurospora crassa Nbp2p orthologues and the high conservation of the Nbp2p binding site in Bck1p orthologues, this function of Nbp2p appears to be conserved across Ascomycetes. Our results also clearly imply a function for the Nbp2p-Ptc1p complex other cellular processes.     

Stu1p is physically associated with beta-tubulin and is required for structural integrity of the mitotic spindle

Formation of the bipolar mitotic spindle relies on a balance of forces acting on the spindle poles. The primary outward force is generated by the kinesin-related proteins of the BimC family that cross-link antiparallel interpolar microtubules and slide them past each other. Here, we provide evidence that Stu1p is also required for the production of this outward force in the yeast Saccharomyces cerevisiae. In the temperature-sensitive stu1-5 mutant, spindle pole separation is inhibited, and preanaphase spindles collapse, with their previously separated poles being drawn together. The temperature sensitivity of stu1-5 can be suppressed by doubling the dosage of Cin8p, a yeast BimC kinesin-related protein. Stu1p was observed to be a component of the mitotic spindle localizing to the midregion of anaphase spindles. It also binds to microtubules in vitro, and we have examined the nature of this interaction. We show that Stu1p interacts specifically with beta-tubulin and identify the domains required for this interaction on both Stu1p and beta-tubulin. Taken together, these findings suggest that Stu1p binds to interpolar microtubules of the mitotic spindle and plays an essential role in their ability to provide an outward force on the spindle poles.     

Yeast Ran-binding protein Yrb1p is required for efficient proteolysis of cell cycle regulatory proteins Pds1p and Sic1p

Ubiquitin-dependent proteolysis of specific target proteins is required for several important steps during the cell cycle. Degradation of such proteins is strictly cell cycle-regulated and triggered by two large ubiquitin ligases, termed anaphase-promoting complex (APC) and Skp1/Cullin/F-box complex (SCF). Here we show that yeast Ran-binding protein 1 (Yrb1p), a predominantly cytoplasmic protein implicated in nucleocytoplasmic transport, is required for cell cycle regulated protein degradation. Depletion of Yrb1p results in the accumulation of unbudded G(1) cells and of cells arrested in mitosis implying a function of Yrb1p in the G(1)/S transition and in the progression through mitosis. Temperature-sensitive yrb1-51 mutants are defective in APC-mediated degradation of the anaphase inhibitor protein Pds1p and in degradation of the cyclin-dependent kinase inhibitor Sic1p, a target of SCF. Thus, Yrb1p is crucial for efficient APC- and SCF-mediated proteolysis of important cell cycle regulatory proteins. We have identified the UBS1 gene as a multicopy suppressor of yrb1-51 mutants. Ubs1p is a nuclear protein, and its deletion is synthetic lethal with a yrb1-51 mutation. Interestingly, UBS1 was previously identified as a multicopy suppressor of cdc34-2 mutants, which are defective in SCF activity. We suggest that Ubs1p may represent a link between nucleocytoplasmic transport and ubiquitin ligase activity.     

Ganoderma lucidum phosphoglucomutase is required for hyphal growth,polysaccharide production,and cell wall integrity

Applied Microbiology and Biotechnology - Phosphoglucomutase (pgm) is an important enzyme in carbohydrate metabolism that is located at the branching point between glycolysis and the Leloir pathway....     

Golgi-localized UDP-glucose transporter is required for cell wall integrity in rice

Cell wall-related nucleotide sugar transporters (NSTs) theoretically supply the cytosolic nucleotide sugars for glycosyltransferases (GTs) to carry out ploysaccharide synthesis and modification in the Golgi apparatus. However, the regulation of cell wall synthesis by NSTs remains undescribed. Recently, we have reported the functional characterization of Oryza sativa nucleotide sugar transport (Osnst1) mutant and its corresponding gene. OsNST1/BC14 is localized in the Golgi apparatus and transports UDP-glucose. This mutant provides us with a unique opportunity for evaluation of its broad impacts on cell wall structure and components. We previously examined cell wall composition of bc14 and wild type plants. Here, the spatial distribution of these cell wall alterations was analyzed by immunolabeling approach. Analysis of the sugar yield in different cell wall fractions indicated that this mutation improves the extractability of cell wall components. Field emission scanning electron microscopy further showed that the orientation of microfibrils in bc14 is irregular when compared to that in wild type. Therefore, this UDP-glucose transporter, making substrates available for polysaccharide biosynthesis, plays a critical role in maintaining cell wall integrity.Key words: UDP-glucose transporter, Golgi apparatus, cell wall polysaccharides, xylan, riceNucleotide sugars mainly generated in cytosol are the substrates for the synthesis of cell wall polysaccharides. Supply of nucleotide sugars is thus a key level for regulation of cell wall components and structure. Mutation in MUR1, an isoform of GDP-D-mannose-4,6-dehydratase, causes reduced amount of GDP-fucose and abnormal xyloglucan structure.^1,2 Disturbance of UDP-rhamnose synthesis via the mutation in RHM2/MUM4 decreases the rhamnogalacturonan I contents in Arabidopsis seeds. Cellulose synthase catalytic subunits (CESAs) generally use cytosolic UDP-glucoses to synthesize cellulose on the plasma membrane. UDP-glucose can be produced either via the catalysis of sucrose by sucrose synthase (SuSy) or through the phosphorylation of glucose-1-phosphate by UDP-glucose pyrophosphorylase (UGPase).³ Suppression of SuSy function in cotton inhibited fiber initiation and elongation.⁴ For the synthesis of noncellulosic polysaccharides occurring inside the Golgi lumen, the cytosolic nucleotide sugars should be translocated inwards by Golgi nucleotide sugar transporters (NSTs).⁵ However, this hypothesis remains to be confirmed, although transport activities have been identified in some plant NSTs.^6–10 Altering the precursor supply may also affect the overall carbon allocation in plants. It is reasonable that substrate regulation often causes pleiotropic effects on cell wall biosynthesis and plant growth. Without genetic resources or mutants on cell wall related NST, the exact evaluation of NSTs'' impacts on cell wall structure and composition is largely delayed. Until recently, we identified a Golgi-localized transporter OsNST1 mutant in rice. This transporter has been found to supply UDP-glucose for the formation of matrix polysaccharides, thereby modulating cellulose biosynthesis.¹¹ Here, we examine these alterations of cell wall polymers at the cellular level. The orientation of cellulose microfibrils and extractability of wall polysaccharides were also compared between the mutant and wild type. All those further our understandings of the functions of NSTs and the synergetic synthesis of different polymers.     

Farnesylation of YDJ1p is required for function at elevated growth temperatures in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae YDJ1 protein (YDJ1p) contains a C-terminal CaaX box motif common to proteins that are modified by prenylation. In the present study we show that YDJ1p is a specific substrate for both yeast and mammalian protein farnesyltransferase enzymes in vitro. A mutant form of YDJ1p, in which the conserved cysteine of the CaaX box is mutated to a serine (ydj1-S406p), cannot be farnesylated in vitro. After expression in S. cerevisiae, ydj1-S406p displays a reduced electrophoretic mobility and an increased cytosolic localization in subcellular fractionation experiments when compared to wild type YDJ1p. Expression of ydj1-S406 in cells lacking YDJ1 results in a temperature-sensitive growth phenotype in S. cerevisiae. These data indicate that farnesylation of YDJ1p is required for its function at elevated temperatures.     

Condensin is required for nonhistone protein assembly and structural integrity of vertebrate mitotic chromosomes

The dramatic condensation of chromosomes that occurs during mitosis is widely thought to be largely controlled by a protein complex termed condensin. Here, we describe a conditional knockout of the condensin subunit ScII/SMC2 in chicken DT40 cells. In cells lacking this condensin subunit, chromosome condensation is delayed, but ultimately reaches near-normal levels. However, these chromosomes are structurally compromised. Kinetochores appear normal, but the localization of nonhistone proteins such as topoisomerase II and INCENP is aberrant. Both proteins also fail to partition into the chromosome scaffold fraction, which appears to be largely missing in the absence of condensin. Furthermore, the chromosomes lack structural integrity, as defined by an assay that tests the stability of the chromosomal higher-order structure. Thus, a major function of condensin is to promote the correct association of nonhistone proteins with mitotic chromosomes, and this is essential for establishment of a robust chromosome structure.     

Nucleophosmin is required for DNA integrity and p19Arf protein stability

Nucleophosmin (NPM) is a nucleolar phosphoprotein that binds the tumor suppressors p53 and p19(Arf) and is thought to be indispensable for ribogenesis, cell proliferation, and survival after DNA damage. The NPM gene is the most frequent target of genetic alterations in leukemias and lymphomas, though its role in tumorigenesis is unknown. We report here the first characterization of a mouse NPM knockout strain. Lack of NPM expression results in accumulation of DNA damage, activation of p53, widespread apoptosis, and mid-stage embryonic lethality. Fibroblasts explanted from null embryos fail to grow and rapidly acquire a senescent phenotype. Transfer of the NPM mutation into a p53-null background rescued apoptosis in vivo and fibroblast proliferation in vitro. Cells null for both p53 and NPM grow faster than control cells and are more susceptible to transformation by activated oncogenes, such as mutated Ras or overexpressed Myc. In the absence of NPM, Arf protein is excluded from nucleoli and is markedly less stable. Our data demonstrate that NPM regulates DNA integrity and, through Arf, inhibits cell proliferation and are consistent with a putative tumor-suppressive function of NPM.     

Saccharomyces cerevisiae Mob1p is required for cytokinesis and mitotic exit

The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved set of genes that mediate the transition from mitosis to G(1) by regulating mitotic cyclin degradation and the inactivation of cyclin-dependent kinase (CDK). Here, we demonstrate that, in addition to mitotic exit, S. cerevisiae MEN gene MOB1 is required for cytokinesis and cell separation. The cytokinesis defect was evident in mob1 mutants under conditions in which there was no mitotic-exit defect. Observation of live cells showed that yeast myosin II, Myo1p, was present in the contractile ring at the bud neck but that the ring failed to contract and disassemble. The cytokinesis defect persisted for several mitotic cycles, resulting in chains of cells with correctly segregated nuclei but with uncontracted actomyosin rings. The cytokinesis proteins Cdc3p (a septin), actin, and Iqg1p/ Cyk1p (an IQGAP-like protein) appeared to correctly localize in mob1 mutants, suggesting that MOB1 functions subsequent to actomyosin ring assembly. We also examined the subcellular distribution of Mob1p during the cell cycle and found that Mob1p first localized to the spindle pole bodies during mid-anaphase and then localized to a ring at the bud neck just before and during cytokinesis. Localization of Mob1p to the bud neck required CDC3, MEN genes CDC5, CDC14, CDC15, and DBF2, and spindle pole body gene NUD1 but was independent of MYO1. The localization of Mob1p to both spindle poles was abolished in cdc15 and nud1 mutants and was perturbed in cdc5 and cdc14 mutants. These results suggest that the MEN functions during the mitosis-to-G(1) transition to control cyclin-CDK inactivation and cytokinesis.     

Phosphorylation of mitotic kinesin-like protein 2 by polo-like kinase 1 is required for cytokinesis

We have investigated the function of mitotic kinesin-like protein (MKlp) 2, a kinesin localized to the central spindle, and demonstrate that its depletion results in a failure of cleavage furrow ingression and cytokinesis, and disrupts localization of polo-like kinase 1 (Plk1). MKlp2 is a target for Plk1, and phosphorylated MKlp2 binds to the polo box domain of Plk1. Plk1 also binds directly to microtubules and targets to the central spindle via its polo box domain, and this interaction controls the activity of Plk1 toward MKlp2. An antibody to the neck region of MKlp2 that prevents phosphorylation of MKlp2 by Plk1 causes a cytokinesis defect when introduced into cells. We propose that phosphorylation of MKlp2 by Plk1 is necessary for the spatial restriction of Plk1 to the central spindle during anaphase and telophase, and the complex of these two proteins is required for cytokinesis.     

Mac1, a fission yeast transmembrane protein localizing to the poles and septum,is required for correct cell separation at high temperatures

Schizosaccharomyces pombe represents a genetic model system for studying cell polarity and division in eukaryotes. We report here the identification of Mac1, a novel fission yeast protein that localized predominantly to the cell tips and septum. Sequences corresponding to roughly the first 180 amino acids of Mac1, which exhibited weak homology to the transmembrane domains of the Aspergillus Pall protein [Mol. Microbiol. 30 (1998) 259], were found to specify localization to the cell periphery. The other 574 amino acids of Mac1 localized to the cytoplasm when expressed alone, thus suggesting that the N-terminal part of Mac1 functions as a plasma membrane anchor for the rest of the protein. In pom1 null mutant cells, which never switch from unipolar to bipolar growth but, instead, grow exclusively at the randomly chosen end [Genes Dev. 12 (1998) 1356], Mac1 was, nevertheless, found at both poles, thus suggesting that Mac1 does not specifically localize to the sites of growth. mac1 null mutant cells had no overt phenotype at 22-32 degrees C, but, nevertheless, displayed a marked decrease in viability at 34-36 degrees C, accompanied by severe separation defects. Overexpression of mac1 resulted in similar defects. Our data suggest that a correct dosage of Mac1 is needed for correct cell separation at elevated temperatures of growth.     

Schizosaccharomyces pombe Hsk1p is a potential cds1p target required for genome integrity

The fission yeast Hsk1p kinase is an essential activator of DNA replication. Here we report the isolation and characterization of a novel mutant allele of the gene. Consistent with its role in the initiation of DNA synthesis, hsk1(ts) genetically interacts with several S-phase mutants. At the restrictive temperature, hsk1(ts) cells suffer abnormal S phase and loss of nuclear integrity and are sensitive to both DNA-damaging agents and replication arrest. Interestingly, hsk1(ts) mutants released to the restrictive temperature after early S-phase arrest in hydroxyurea (HU) are able to complete bulk DNA synthesis but they nevertheless undergo an abnormal mitosis. These findings indicate a second role for hsk1 subsequent to HU arrest. Consistent with a later S-phase role, hsk1(ts) is synthetically lethal with Deltarqh1 (RecQ helicase) or rad21ts (cohesin) mutants and suppressed by Deltacds1 (RAD53 kinase) mutants. We demonstrate that Hsk1p undergoes Cds1p-dependent phosphorylation in response to HU and that it is a direct substrate of purified Cds1p kinase in vitro. These results indicate that the Hsk1p kinase is a potential target of Cds1p regulation and that its activity is required after replication initiation for normal mitosis.     

Pkh1 and Pkh2 differentially phosphorylate and activate Ypk1 and Ykr2 and define protein kinase modules required for maintenance of cell wall integrity

Saccharomyces cerevisiae Pkh1 and Pkh2 are functionally redundant homologs of mammalian protein kinase, phosphoinositide-dependent protein kinase-1. They activate two closely related, functionally redundant enzymes, Ypk1 and Ykr2 (homologs of mammalian protein kinase, serum- and glucocorticoid-inducible protein kinase). We found that Ypk1 has a more prominent role than Ykr2 in mediating their shared essential function. Considerable evidence demonstrated that Pkh1 preferentially activates Ypk1, whereas Pkh2 preferentially activates Ykr2. Loss of Pkh1 (but not Pkh2) reduced Ypk1 activity; conversely, Pkh1 overexpression increased Ypk1 activity more than Pkh2 overexpression. Loss of Pkh2 reduced Ykr2 activity; correspondingly, Pkh2 overexpression increased Ykr2 activity more than Pkh1 overexpression. When overexpressed, a catalytically active C-terminal fragment (kinase domain) of Ypk1 was growth inhibitory; loss of Pkh1 (but not Pkh2) alleviated toxicity. Loss of Pkh2 (but not Pkh1) exacerbated the slow growth phenotype of a ypk1Delta strain. This Pkh1-Ypk1 and Pkh2-Ykr2 dichotomy is not absolute because all double mutants (pkh1Delta ypk1Delta, pkh2Delta ypk1Delta, pkh1Delta ykr2Delta, and pkh2Delta ykr2Delta) were viable. Compartmentation contributes to selectivity because Pkh1 and Ypk1 were located exclusively in the cytosol, whereas Pkh2 and Ykr2 entered the nucleus. At restrictive temperature, ypk1-1(ts) ykr2Delta cells lysed rapidly, but not in medium containing osmotic support. Dosage and extragenic suppressors were selected. Overexpression of Exg1 (major exoglucanase), or loss of Kex2 (endoprotease involved in Exg1 processing), rescued growth at high temperature. Viability was also maintained by PKC1 overexpression or an activated allele of the downstream protein kinase (BCK1-20). Conversely, absence of Mpk1 (distal mitogen-activated protein kinase of the PKC1 pathway) was lethal in ypk1-1(ts) ykr2Delta cells. Thus, Pkh1-Ypk1 and Pkh2-Ykr2 function in a novel pathway for cell wall integrity that acts in parallel with the Pkc1-dependent pathway.