Direct activation of the fission yeast PAK Shk1 by the novel SH3 domain protein, Skb5

The p21-activated kinase (PAK) homolog Shk1 is essential for cell viability in the fission yeast Schizosaccharomyces pombe. Roles have been established for Shk1 in the regulation of cell morphology, sexual differentiation, and mitosis in S. pombe. In this report, we describe the genetic and molecular characterization of a novel SH3 domain protein, Skb5, identified as a result of a two-hybrid screen for Shk1 interacting proteins. S. pombe cells carrying a deletion of the skb5 gene exhibit no discernible phenotypic defects under normal growth conditions, but when subjected to hypertonic stress, become spheroidal in shape and growth impaired. Both of these defects can be suppressed by overexpression of the Shk1 modulator, Skb1. The growth inhibition that results from overexpression of Shk1 in S. pombe cells is markedly suppressed by a null mutation in the skb5 gene, suggesting that Skb5 contributes positively to the function of Shk1 in vivo. Consistent with this notion, we show that Skb5 stimulates Shk1 catalytic function in S. pombe cells. Furthermore, and perhaps most significantly, we show that bacterially expressed recombinant Skb5 protein directly stimulates the catalytic activity of recombinant Shk1 kinase in vitro. These and additional data described herein demonstrate that Skb5 is a direct activator of Shk1 in fission yeast.     

The highly conserved protein methyltransferase, Skb1, is a mediator of hyperosmotic stress response in the fission yeast Schizosaccharomyces pombe

The p21-activated kinase, Shk1, is required for cell viability, establishment and maintenance of cell polarity, and proper mating response in the fission yeast, Schizosaccharomyces pombe. Previous genetic studies suggested that a presumptive protein methyltransferase, Skb1, functions as a positive modulator of Shk1. However, unlike Shk1, Skb1 is not required for viability or mating of S. pombe cells and contributes only modestly to the regulation of cell morphology under normal growth conditions. Here we demonstrate that Skb1 plays a more significant role in regulating cell growth and polarity under conditions of hyperosmotic stress. We provide evidence that the inability of skb1Delta cells to properly maintain cell polarity in hyperosmotic conditions results from inefficient subcellular targeting of F-actin. We show that Skb1 localizes to cell ends, sites of septation, and nuclei of S. pombe cells. Hyperosmotic shock results in substantial delocalization of Skb1 from cell ends and nuclei, as well as stimulation of Skb1 protein methyltransferase activity. Taken together, our results demonstrate a new role for Skb1 as a mediator of hyperosmotic stress response in fission yeast. We show that the protein methyltransferase activity of the human Skb1 homolog, Skb1Hs, is also stimulated by hyperosmotic stress in fission yeast, providing evidence for evolutionary conservation of a role for Skb1-related proteins as mediators of hyperosmotic stress response, as well as mechanisms involved in regulating this novel class of protein methyltransferases.     

Control of cell polarity in fission yeast by association of Orb6p kinase with the highly conserved protein methyltransferase Skb1p

In the fission yeast Schizosaccharomyces pombe, proper establishment and maintenance of cell polarity require Orb6p, a highly conserved serine/threonine kinase involved in regulating both cell morphogenesis and cell cycle control. Orb6p localizes to the cell tips during interphase and to the cell septum during mitosis. To investigate the mechanisms involved in Orb6p function, we conducted a two-hybrid screen to identify proteins that interact with Orb6p. Using this approach, we identified Skb1p, a highly conserved protein methyltransferase that has been implicated previously in cell cycle control, in the coordination of cell cycle progression with morphological changes, and in hyperosmotic stress response. We found that Skb1p associates with Orb6p in S. pombe cells and that the two proteins interact directly in vitro. Loss of Skb1p exacerbates the phenotype of orb6 mutants, suggesting that Skb1p and Orb6p functionally interact in S. pombe cells. Our results suggest that Skb1p affects the intracellular localization of Orb6p and that loss of Skb1p leads to a redistribution of the Orb6p kinase away from the cell tips. Furthermore, we found that Orb6p kinase activity is strongly increased following exposure to salt shock, suggesting that Orb6p has a role in cell response to hyperosmotic stress. Previous studies have shown that Skb1p interacts with the fission yeast p21-activated kinase homologue Pak1p/Shk1p to regulate cell polarity and cell cycle progression. Our findings identify Orb6p as an additional target for Skb1p and suggest a novel function for Skb1p in the control of cell polarity by regulating the subcellular localization of Orb6p.     

Mis-specification of cortical identity in a fission yeast PAK mutant

The regulation of cell polarity in the fission yeast Schizosaccharomyces pombe is apparent in the restriction of extensile growth to the two ends of a cylindrically shaped cell, and in a specific transition - termed 'new-end take-off' (NETO) - between monopolar and bipolar growth mid-way through the cell cycle [1]. Several genes have been identified that affect one or more aspects of cell polarity (reviewed in [2] [3]), and the molecular pathways regulating cell polarity in fission yeast appear to be conserved among eukaryotes [3] [4] [5] [6] [7] [8] [9], but it is less clear how the proteins involved organize polarity at the level of the entire cell. Here, we describe novel cytological markers of cell polarity in fission yeast and their unusual localization in the monopolar growth mutant orb2-34, which carries a non-lethal mutation in the essential gene shk1(+)/pak1(+)/orb2(+), which encodes a p21-activated kinase (PAK) family member [8] [9] [10] [11] [12]. Our results suggest that, in contrast to other monopolar-growing mutants, the monopolar phenotype of the orb2-34 mutant might not be due to a defect in activating end growth per se, but rather reflects a failure of one of the cell ends to maintain the molecular properties that identify an end. Thus, one role of the Shk1/Pak1 kinase in vivo might be to contribute to how a cell recognizes its ends as sites for growth.     

PNG1, a yeast gene encoding a highly conserved peptide:N-glycanase

It has been proposed that cytoplasmic peptide:N-glycanase (PNGase) may be involved in the proteasome-dependent quality control machinery used to degrade newly synthesized glycoproteins that do not correctly fold in the ER. However, a lack of information about the structure of the enzyme has limited our ability to obtain insight into its precise biological function. A PNGase-defective mutant (png1-1) was identified by screening a collection of mutagenized strains for the absence of PNGase activity in cell extracts. The PNG1 gene was mapped to the left arm of chromosome XVI by genetic approaches and its open reading frame was identified. PNG1 encodes a soluble protein that, when expressed in Escherichia coli, exhibited PNGase activity. PNG1 may be required for efficient proteasome-mediated degradation of a misfolded glycoprotein. Subcellular localization studies indicate that Png1p is present in the nucleus as well as the cytosol. Sequencing of expressed sequence tag clones revealed that Png1p is highly conserved in a wide variety of eukaryotes including mammals, suggesting that the enzyme has an important function.     

The kelch repeat protein,Tea1, is a potential substrate target of the p21-activated kinase,Shk1, in the fission yeast,Schizosaccharomyces pombe

The p21-activated kinase (PAK) homolog, Shk1, is a critical component of a multifunctional Ras/Cdc42/PAK complex required for viability, polarized growth and cell shape, and sexual differentiation in the fission yeast, Schizosaccharomyces pombe. Substrate targets of the Shk1 kinase have not previously been described. Here we show that the S. pombe cell polarity factor, Tea1, is directly phosphorylated by Shk1 in vitro. We demonstrate further that Tea1 is phosphorylated in S. pombe cells and that its level of phosphorylation is significantly reduced in cells defective in Shk1 function. Consistent with a role for Tea1 as a potential downstream effector of Shk1, we show that a tea1 null mutation rescues the Shk1 hyperactivity-induced lethal phenotype caused by loss of function of the essential Shk1 inhibitor, Skb15. All phenotypes associated with Skb15 loss, including defects in actin cytoskeletal organization, chromosome segregation, and cytokinesis, are suppressed by tea1 Delta, suggesting that Tea1 is a potential mediator of multiple Shk1 functions. S. pombe cells carrying a weak hypomorphic allele of shk1 together with a tea1 Delta mutation exhibit a cytokinesis defective phenotype that is significantly more severe than that observed in the respective single mutants, providing evidence that Shk1 and Tea1 cooperate to regulate cytokinesis. In addition, we show that S. pombe cells carrying the orb2-34 allele of shk1 exhibit a pattern of monopolar growth similar to that observed in tea1 Delta cells, suggesting that Shk1 and Tea1 may regulate one or more common processes involved in the regulation of polarized cell growth. Taken together, our results strongly implicate Tea1 as a potential substrate-effector of the Shk1 kinase.     

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.     

Genetic evidence for phospholipid-mediated regulation of the Rab GDP-dissociation inhibitor in fission yeast

We have previously identified mutant alleles of genes encoding two Rab proteins, Ypt3 and Ryh1, through a genetic screen using the immunosuppressant drug FK506 in fission yeast. In the same screen, we isolated gdi1-i11, a mutant allele of the essential gdi1+ gene encoding Rab GDP-dissociation inhibitor. In gdi1-i11, a conserved Gly267 was substituted by Asp. The Gdi1G267D protein failed to extract Rabs from membrane and Rabs were depleted from the cytosolic fraction in the gdi1-i11 mutant cells. Consistently, the Gdi1G267D protein was found mostly in the membrane fraction, whereas wild-type Gdi1 was found in both the cytosolic and the membrane fraction. Notably, overexpression of spo20+, encoding a phosphatidylcholine/phosphatidylinositol transfer protein, rescued gdi1-i11 mutation, but not ypt3-i5 or ryh1-i6. The gdi1-i11 and spo20-KC104 mutations are synthetically lethal, and the wild-type Gdi1 failed to extract Rabs from the membrane in the spo20-KC104 mutant. The phosphatidylinositol-transfer activity of Spo20 is dispensable for the suppression of the gdi1-i11 mutation, suggesting that the phosphatidylcholine-transfer activity is important for the suppression. Furthermore, knockout of the pct1+ gene encoding a choline phosphate cytidyltransferase rescued the gdi1-i11 mutation. Together, our findings suggest that Spo20 modulates Gdi1 function via regulation of phospholipid metabolism of the membranes.     

rum1: a CDK inhibitor regulating G1 progression in fission yeast

In all eukaryotes, entry into mitosis from G2 phase is initiated by a complex of the cdc2 kinase and a B-type cyclin. It has now been shown that, in fission yeast, B-type cyclins also activate cdc2 in G1, thus governing cell-cycle commitment, as well as the onset of S phase. In this article, Karim Labib and Sergio Moreno review the evidence that ruml inhibits the kinase activity of cdc2 associated with B-type cyclins and is an important regulator o f G1 progression in fission yeast.     

The p21-activated protein kinase inhibitor Skb15 and its budding yeast homologue are 60S ribosome assembly factors

Ribosome biogenesis is driven by a large number of preribosomal factors that associate with and dissociate from the preribosomal particles along the maturation pathway. We have previously shown that budding yeast Mak11, whose homologues in other eukaryotes were described as modulating a p21-activated protein kinase function, accumulates in Rlp24-associated pre-60S complexes when their maturation is impeded in Saccharomyces cerevisiae. The functional inactivation of WD40 repeat protein Mak11 interfered with the 60S rRNA maturation, led to a cell cycle delay in G(1), and blocked green fluorescent protein-tagged Rpl25 in the nucleoli of yeast cells, indicating an early role of Mak11 in ribosome assembly. Surprisingly, Mak11 inactivation also led to a dramatic destabilization of Rlp24. The suppression of the thermosensitive phenotype of a mak11 mutant by RLP24 overexpression and a direct in vitro interaction between Rlp24 and Mak11 suggest that Mak11 acts as an Rlp24 cofactor during early steps of 60S ribosomal subunit assembly. Moreover, we found that Skb15, the Mak11 homologue in Schizosaccharomyces pombe, also associated with preribosomes and affected 60S biogenesis in fission yeast. It is thus likely that the previously observed phenotypes for MAK11 homologues in other eukaryotes are secondary to the main function of these proteins in ribosome formation.     

Dnt1 acts as a mitotic inhibitor of the spindle checkpoint protein dma1 in fission yeast

The Schizosaccharomyces pombe checkpoint protein Dma1 couples mitotic progression with cytokinesis and is important in delaying mitotic exit and cytokinesis when kinetochores are not properly attached to the mitotic spindle. Dma1 is a ubiquitin ligase and potential functional relative of the human tumor suppressor Chfr. Dma1 delays mitotic exit and cytokinesis by ubiquitinating a scaffold protein (Sid4) of the septation initiation network, which, in turn, antagonizes the ability of the Polo-like kinase Plo1 to promote cell division. Here we identify Dnt1 as a Dma1-binding protein. Several lines of evidence indicate that Dnt1 inhibits Dma1 function during metaphase. First, Dnt1 interacts preferentially with Dma1 during metaphase. Second, Dma1 ubiquitin ligase activity and Sid4 ubiquitination are elevated in dnt1 cells. Third, the enhanced mitotic defects in dnt1Δ plo1 double mutants are partially rescued by deletion of dma1(+), suggesting that the defects in dnt1 plo1 double mutants are attributable to excess Dma1 activity. Taken together, these data show that Dnt1 acts to restrain Dma1 activity in early mitosis to allow normal mitotic progression.     

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.     

Architecture of the budding yeast kinetochore reveals a conserved molecular core

How kinetochore proteins are organized to connect chromosomes to spindle microtubules, and whether any structural and organizational themes are common to kinetochores from distantly related organisms, are key unanswered questions. Here, we used affinity chromatography and mass spectrometry to generate a map of kinetochore protein interactions. The budding yeast CENP-C homologue Mif2p specifically copurified with histones H2A, H2B, and H4, and with the histone H3-like CENP-A homologue Cse4p, strongly suggesting that Cse4p replaces histone H3 in a specialized centromeric nucleosome. A novel four-protein Mtw1 complex, the Nnf1p subunit of which has homology to the vertebrate kinetochore protein CENP-H, also copurified with Mif2p and a variety of central kinetochore proteins. We show that Mif2 is a critical in vivo target of the Aurora kinase Ipl1p. Chromatin immunoprecipitation studies demonstrated the biological relevance of these associations. We propose that a molecular core consisting of CENP-A, -C, -H, and Ndc80/HEC has been conserved from yeast to humans to link centromeres to spindle microtubules.     

Genetic and biochemical analyses of Pfh1 DNA helicase function in fission yeast

The Schizosaccharomyces pombe pfh1⁺ gene (PIF1 homolog) encodes an essential enzyme that has both DNA helicase and ATPase activities and is implicated in lagging strand DNA processing. Mutations in the pfh1⁺ gene suppress a temperature-sensitive allele of cdc24⁺, which encodes a protein that functions with Schizosaccharomyces pombe Dna2 in Okazaki fragment processing. In this study, we describe the enzymatic properties of the Pfh1 helicase and the genetic interactions between pfh1 and cdc24, dna2, cdc27 or pol 3, all of which are involved in the Okazaki fragment metabolism. We show that a full-length Pfh1 fusion protein is active as a monomer. The helicase activity of Pfh1 displaced only short (<30 bp) duplex DNA regions efficiently in a highly distributive manner and was markedly stimulated by the presence of a replication-fork-like structure in the substrate. The temperature-sensitive phenotype of a dna2-C2 or a cdc24-M38 mutant was suppressed by pfh1-R20 (a cold-sensitive mutant allele of pfh1) and overexpression of wild-type pfh1⁺ abolished the ability of the pfh1 mutant alleles to suppress dna2-C2 and cdc24-M38. Purified Pfh1-R20 mutant protein displayed significantly reduced ATPase and helicase activities. These results indicate that the simultaneous loss-of-function mutations of pfh1⁺ and dna2⁺ (or cdc24⁺) are essential to restore the growth defect. Our genetic data indicate that the Pfh1 DNA helicase acts in concert with Cdc24 and Dna2 to process single-stranded DNA flaps generated in vivo by pol δ-mediated lagging strand displacement DNA synthesis.     

Pgt1, a glutathione transporter from the fission yeast Schizosaccharomyces pombe

The Schizosaccharomyces pombe ORF, SPAC29B12.10c, a predicted member of the oligopeptide transporter (OPT) family, was identified as a gene encoding the S. pombe glutathione transporter ( Pgt1 ) by a genetic strategy that exploited the requirement of the cys1a Δ strain of S. pombe (which is defective in cysteine biosynthesis) for either cysteine or glutathione, for growth. Disruption of the ORF in the cys1a Δ strain led to an inability to grow on glutathione as a source of cysteine. Cloning and subsequent biochemical characterization of the ORF revealed that a high-affinity transporter for glutathione ( K _m=63 μM) that was found to be localized to the plasma membrane. The transporter was specific for glutathione, as significant inhibition in glutathione uptake could be observed only by either reduced or oxidized glutathione, or glutathione conjugates, but not by dipeptides or tripeptides. Furthermore, although glu–cys–gly, an analogue of glutathione (γ-glu–cys–gly), could be utilized as a sulphur source, the growth was not Pgt1 dependent. This further underlined the specificity of this transporter for glutathione. The strong repression of pgt1⁺ expression by cysteine suggested a role in scavenging glutathione from the extracellular environment for the maintenance of sulphur homeostasis in this yeast.