Feedback Regulation of SIN by Etd1 and Rho1 in Fission Yeast

In fission yeast, the septation initiation network (SIN) is thought to promote cytokinesis by downstream activation of Rho1, a conserved GTPase that controls cell growth and division. Here we show that Etd1 and PP2A-Pab1, antagonistic regulators of SIN, are Rho1 regulators. Our genetic and biochemical studies indicate that a C-terminal region of Etd1 may activate Rho1 by directly binding it, whereas an N-terminal domain confers its ability to localize at the growing tips and the division site where Rho1 functions. In opposition to Etd1, our results indicate that PP2A-Pab1 inhibits Rho1. The SIN cascade is upstream-regulated by the Spg1 GTPase. In the absence of Etd1, activity of Spg1 drops down prematurely, thereby inactivating SIN. Interestingly, we find that ectopic activation of Rho1 restores Spg1 activity in Etd1-depleted cells. By using a cytokinesis block strategy, we show that Rho1 is essential to feedback-activate Spg1 during actomyosin ring constriction. Therefore, activation of Spg1 by Rho1, which in turn is regulated by Etd1, uncovers a novel feedback loop mechanism that ensures SIN activity while cytokinesis is progressing.     

Roles of the TRAPP-II Complex and the Exocyst in Membrane Deposition during Fission Yeast Cytokinesis

The cleavage-furrow tip adjacent to the actomyosin contractile ring is believed to be the predominant site for plasma-membrane insertion through exocyst-tethered vesicles during cytokinesis. Here we found that most secretory vesicles are delivered by myosin-V on linear actin cables in fission yeast cytokinesis. Surprisingly, by tracking individual exocytic and endocytic events, we found that vesicles with new membrane are deposited to the cleavage furrow relatively evenly during contractile-ring constriction, but the rim of the cleavage furrow is the main site for endocytosis. Fusion of vesicles with the plasma membrane requires vesicle tethers. Our data suggest that the transport particle protein II (TRAPP-II) complex and Rab11 GTPase Ypt3 help to tether secretory vesicles or tubulovesicular structures along the cleavage furrow while the exocyst tethers vesicles at the rim of the division plane. We conclude that the exocyst and TRAPP-II complex have distinct localizations at the division site, but both are important for membrane expansion and exocytosis during cytokinesis.     

An InCytes from MBC Selection: Roles of Formin Nodes and Myosin Motor Activity in Mid1p-dependent Contractile-Ring Assembly during Fission Yeast Cytokinesis

Two prevailing models have emerged to explain the mechanism of contractile-ring assembly during cytokinesis in the fission yeast Schizosaccharomyces pombe: the spot/leading cable model and the search, capture, pull, and release (SCPR) model. We tested some of the basic assumptions of the two models. Monte Carlo simulations of the SCPR model require that the formin Cdc12p is present in >30 nodes from which actin filaments are nucleated and captured by myosin-II in neighboring nodes. The force produced by myosin motors pulls the nodes together to form a compact contractile ring. Live microscopy of cells expressing Cdc12p fluorescent fusion proteins shows for the first time that Cdc12p localizes to a broad band of 30–50 dynamic nodes, where actin filaments are nucleated in random directions. The proposed progenitor spot, essential for the spot/leading cable model, usually disappears without nucleating actin filaments. α-Actinin ain1 deletion cells form a normal contractile ring through nodes in the absence of the spot. Myosin motor activity is required to condense the nodes into a contractile ring, based on slower or absent node condensation in myo2-E1 and UCS rng3-65 mutants. Taken together, these data provide strong support for the SCPR model of contractile-ring formation in cytokinesis.     

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.     

Phosphatidylethanolamine Is Required for Normal Cell Morphology and Cytokinesis in the Fission Yeast Schizosaccharomyces pombe

To investigate the contributions of phosphatidylethanolamine to the growth and morphogenesis of the fission yeast Schizosaccharomyces pombe, we have characterized three predicted genes in this organism, designated psd1, psd2, and psd3, encoding phosphatidylserine decarboxylases, which catalyze the conversion of phosphatidylserine to phosphatidylethanolamine in both eukaryotic and prokaryotic organisms. S. pombe mutants carrying deletions in any one or two psd genes are viable in complex rich medium and synthetic defined minimal medium. However, mutants carrying deletions in all three psd genes (psd1-3Δ mutants) grow slowly in rich medium and are inviable in minimal medium, indicating that the psd1 to psd3 gene products share overlapping essential cellular functions. Supplementation of growth media with ethanolamine, which can be converted to phosphatidylethanolamine by the Kennedy pathway, restores growth to psd1-3Δ cells in minimal medium, indicating that phosphatidylethanolamine is essential for S. pombe cell growth. psd1-3Δ cells produce lower levels of phosphatidylethanolamine than wild-type cells, even in medium supplemented with ethanolamine, indicating that the Kennedy pathway can only partially compensate for the loss of phosphatidylserine decarboxylase activity in S. pombe. psd1-3Δ cells appear morphologically indistinguishable from wild-type S. pombe cells in medium supplemented with ethanolamine, but when cultured in nonsupplemented medium, they produce high frequencies of abnormally shaped cells as well as cells exhibiting severe septation defects, including multiple, mispositioned, deformed, and misoriented septa. Our results demonstrate that phosphatidylethanolamine is essential for cell growth and for normal cytokinesis and cellular morphogenesis in S. pombe, and they illustrate the usefulness of this model eukaryote for investigating potentially conserved biological and molecular functions of phosphatidylethanolamine.Phosphatidylethanolamine (PE) is a major phospholipid component of cell membranes in both prokaryotic and eukaryotic organisms (34, 35). There are three distinct pathways for PE synthesis in eukaryotic cells: (i) decarboxylation of phosphatidylserine (PS) via reactions catalyzed by PS decarboxylase (PSD) enzymes; (ii) the CDP-ethanolamine branch of the Kennedy pathway, which converts ethanolamine to PE (34); and (iii) acylation of lysophosphatidylethanolamine (21, 29), a reaction that in the budding yeast Saccharomyces cerevisiae is catalyzed by the enzyme Ale1 (22). Genetic studies have demonstrated that PE is essential for cell viability in S. cerevisiae, although the minimal threshold of PE required for cell growth in this organism can apparently be provided by any of the routes of PE synthesis listed above (22). In contrast, the results of mouse knockout experiments indicate that both PSD- and Kennedy pathway-catalyzed pathways for PE synthesis are essential for embryonic development (9, 28, 35).While PE is present in most, if not all, eukaryotic cell membranes, it is particularly enriched in the membranes of mitochondria (32, 35, 37). Indeed, S. cerevisiae mutants carrying a null mutation in the PSD1 gene, which encodes a mitochondrially localized PSD, exhibit phenotypes indicative of mitochondrial dysfunction, as do cells derived from mouse embryos carrying a disruption of the Psid gene, which encodes a protein highly homologous in structure to S. cerevisiae Psd1 (28, 32). A second PSD enzyme in S. cerevisiae, encoded by the PSD2 gene, is localized to Golgi and vacuolar membranes (33, 37). Consistent with a role in vacuolar function, PE has been implicated in the process of autophagy by genetic studies utilizing S. cerevisiae vacuolar targeting mutants and by studies showing that Atg8, a ubiquitin-like protein required for yeast autophagy, is conjugated to PE, as are several related mammalian proteins (19, 20, 27).Interestingly, studies utilizing a streptavidin-conjugated form of the PE-binding peptide cinnamycin demonstrated that PE is enriched at cell division sites in S. cerevisiae, the fission yeast Schizosaccharomyces pombe, and mammalian cells (6, 11). Moreover, streptavidin-conjugated cinnamycin was shown to inhibit the disassembly of the contractile ring and the completion of cytokinesis in cultures of Chinese hamster ovary cells, and a PE-deficient cell line from the same species was found to arrest growth in cytokinesis with an intact contractile ring (7). PE has also been shown to be enriched at the growing ends of interphase S. pombe cells and at the emerging bud cortex in dividing cells of S. cerevisiae, findings that implicate PE in processes controlling polarized cell growth (11).Although S. pombe mutants defective in enzymes that directly catalyze PE synthesis have not been described previously, we recently showed that mutants carrying a null mutation in the PS synthase gene pps1 are ethanolamine auxotrophs that exhibit severe morphology- and cytokinesis-defective phenotypes under ethanolamine-limited growth conditions (17). These findings implicated PE in the regulation of cellular morphogenesis and cytokinesis in S. pombe. To investigate the biological functions of PE in S. pombe, in particular its contributions to the control of cell morphology and cytokinesis, we have in the present study generated and characterized mutants carrying null mutations in three open reading frames predicted to encode PSD enzymes in this organism. In this paper, we describe the phenotypes of S. pombe PSD-null mutants, which demonstrate central roles for PE in the regulation of cell morphology and cytokinesis in this model eukaryote.     

Two Fission Yeast Rab7 Homologs, Ypt7 and Ypt71, Play Antagonistic Roles in the Regulation of Vacuolar Morphology

Small guanine triphosphatases (GTPases) of the Rab family are key regulators of membrane trafficking events between the various subcellular compartments in eukaryotic cells. Rab7 is a conserved protein required in the late endocytic pathway and in lysosome biogenesis. A Schizosaccharomyces pombe ( S. pombe ) homolog of Rab7, Ypt7, is necessary for trafficking from the endosome to the vacuole and for homotypic vacuole fusion. Here, we identified and characterized a second fission yeast Rab7 homolog, Ypt71. Ypt71 is localized to the vacuolar membrane. Cells deleted for ypt71 ⁺ exhibit normal growth rates and morphology. Interestingly, a ypt71 null mutant contains large vacuoles in contrast with the small fragmented vacuoles found in the ypt7 null mutant. Furthermore, the ypt71 mutation does not enhance or alleviate the temperature sensitivity or vacuole fusion defect of ypt7 Δ cells. Like ypt7 Δ cells, overexpression of ypt71 ⁺ caused fragmentation of vacuoles and inhibits vacuole fusion under hypotonic conditions. Thus, the two S. pombe Rab7 homologs act antagonistically in regulating vacuolar morphology. Analysis of a chimeric Ypt7/Ypt71 protein showed that Rab7-directed vacuole dynamics, fusion versus fission, largely depends on the medial region of the protein, including a part of RabSF3/α3-L7.     

Hof1 and Rvs167 Have Redundant Roles in Actomyosin Ring Function during Cytokinesis in Budding Yeast

The Hof1 protein (Homologue of Fifteen) regulates formation of the primary septum during cytokinesis in the budding yeast Saccharomyces cerevisiae, whereas the orthologous Cdc15 protein in fission yeast regulates the actomyosin ring by using its F-BAR domain to recruit actin nucleators to the cleavage site. Here we show that budding yeast Hof1 also contributes to actin ring assembly in parallel with the Rvs167 protein. Simultaneous deletion of the HOF1 and RVS167 genes is lethal, and cells fail to assemble the actomyosin ring as they progress through mitosis. Although Hof1 and Rvs167 are not orthologues, they both share an analogous structure, with an F-BAR or BAR domain at the amino terminus, capable of inducing membrane curvature, and SH3 domains at the carboxyl terminus that bind to specific proline-rich targets. The SH3 domain of Rvs167 becomes essential for assembly of the actomyosin ring in cells lacking Hof1, suggesting that it helps to recruit a regulator of the actin cytoskeleton. This new function of Rvs167 appears to be independent of its known role as a regulator of the Arp2/3 actin nucleator, as actin ring assembly is not abolished by the simultaneous inactivation of Hof1 and Arp2/3. Instead we find that recruitment to the bud-neck of the Iqg1 actin regulator is defective in cells lacking Hof1 and Rvs167, though future studies will be needed to determine if this reflects a direct interaction between these factors. The redundant role of Hof1 in actin ring assembly suggests that the mechanism of actin ring assembly has been conserved to a greater extent across evolution than anticipated previously.     

Reprogramming Cdr2-Dependent Geometry-Based Cell Size Control in Fission Yeast

1. Download high-res image (262KB)
2. Download full-size image

     

A Molecular Evolution Approach to Study the Roles of Tropomyosin in Fission Yeast

Tropomyosin, a coiled-coil protein that binds along the length of the actin filament, is a universal regulator of the actin cytoskeleton. We have taken a bioinformatics/proteomic approach to studying structure-function relationships in this protein. The presence of a single, essential tropomyosin gene, cdc8, in fission yeast, Schizosaccharomyces pombe, enables a systems-based approach to define the residues that are important for cellular functions. Using molecular evolution methodologies we identified the most conserved residues and related them to the coiled coil structure. Mutants in which one or more of 21 of the most conserved surface residues was mutated to Ala were tested for the ability to rescue growth of a temperature-sensitive cdc8 mutant when overexpressed at the restrictive temperature. Based on altered morphology of the septum and actin cytoskeleton, we selected three sets of mutations for construction of mutant cdc8 strains using marker reconstitution mutagenesis and analysis of recombinant protein in vitro: D16A.K30A, V114S.E117A.H118A and R121A.D131A.E138A. The mutations have sequence-specific effects on cellular morphology including cell length, organization of cytoskeletal structures (actin patches, actin cables and contractile rings), and in vitro actin affinity, lending credence to the proteomic approach introduced here. We propose that bioinformatics is a valid analysis tool for defining structure-function relationships in conserved proteins in this model organism.     

UCS Protein Rng3p Is Essential for Myosin-II Motor Activity during Cytokinesis in Fission Yeast

UCS proteins have been proposed to operate as co-chaperones that work with Hsp90 in the de novo folding of myosin motors. The fission yeast UCS protein Rng3p is essential for actomyosin ring assembly and cytokinesis. Here we investigated the role of Rng3p in fission yeast myosin-II (Myo2p) motor activity. Myo2p isolated from an arrested rng3-65 mutant was capable of binding actin, yet lacked stability and activity based on its expression levels and inactivity in ATPase and actin filament gliding assays. Myo2p isolated from a myo2-E1 mutant (a mutant hyper-sensitive to perturbation of Rng3p function) showed similar behavior in the same assays and exhibited an altered motor conformation based on limited proteolysis experiments. We propose that Rng3p is not required for the folding of motors per se, but instead works to ensure the activity of intrinsically unstable myosin-II motors. Rng3p is specific to conventional myosin-II and the actomyosin ring, and is not required for unconventional myosin motor function at other actin structures. However, artificial destabilization of myosin-I motors at endocytic actin patches (using a myo1-E1 mutant) led to recruitment of Rng3p to patches. Thus, while Rng3p is specific to myosin-II, UCS proteins are adaptable and can respond to changes in the stability of other myosin motors.     

HIV-1 Protease in the Fission Yeast Schizosaccharomyces pombe

Background

HIV-1 protease (PR) is an essential viral enzyme. Its primary function is to proteolyze the viral Gag-Pol polyprotein for production of viral enzymes and structural proteins and for maturation of infectious viral particles. Increasing evidence suggests that PR cleaves host cellular proteins. However, the nature of PR-host cellular protein interactions is elusive. This study aimed to develop a fission yeast (Schizosaccharomyces pombe) model system and to examine the possible interaction of HIV-1 PR with cellular proteins and its potential impact on cell proliferation and viability.

Results

A fission yeast strain RE294 was created that carried a single integrated copy of the PR gene in its chromosome. The PR gene was expressed using an inducible nmt1 promoter so that PR-specific effects could be measured. HIV-1 PR from this system cleaved the same indigenous viral p6/MA protein substrate as it does in natural HIV-1 infections. HIV-1 PR expression in fission yeast cells prevented cell proliferation and induced cellular oxidative stress and changes in mitochondrial morphology that led to cell death. Both these PR activities can be prevented by a PR-specific enzymatic inhibitor, indinavir, suggesting that PR-mediated proteolytic activities and cytotoxic effects resulted from enzymatic activities of HIV-1 PR. Through genome-wide screening, a serine/threonine kinase, Hhp2, was identified that suppresses HIV-1 PR-induced protease cleavage and cell death in fission yeast and in mammalian cells, where it prevented PR-induced apoptosis and cleavage of caspase-3 and caspase-8.

Conclusions

This is the first report to show that HIV-1 protease is functional as an enzyme in fission yeast, and that it behaves in a similar manner as it does in HIV-1 infection. HIV-1 PR-induced cell death in fission yeast could potentially be used as an endpoint for mechanistic studies, and this system could be used for developing a high-throughput system for drug screenings.     

裂殖酵母Pap1应答H2O2氧化胁迫的机制

转录因子Pap1是裂殖酵母(Schizosaccharomyces pombe)应答H2O2氧化胁迫反应中的关键调控因子.高浓度的H2O2激活蛋白激酶Sty1途径清除过量的H2O2,使H2O2降至较低浓度再活化Pap1;低浓度的则直接氧化活化Pap1,导致Pap1快速向细胞核内运输从而激活Pap1相关基因的表达.本文综述了裂殖酵母中转录因子Pap1在不同浓度H2O2胁迫下的激活途径,以及蛋白激酶Sty1对Pap1激活的重要作用     

Ingression Progression Complexes Control Extracellular Matrix Remodelling during Cytokinesis in Budding Yeast

Eukaryotic cells must coordinate contraction of the actomyosin ring at the division site together with ingression of the plasma membrane and remodelling of the extracellular matrix (ECM) to support cytokinesis, but the underlying mechanisms are still poorly understood. In eukaryotes, glycosyltransferases that synthesise ECM polysaccharides are emerging as key factors during cytokinesis. The budding yeast chitin synthase Chs2 makes the primary septum, a special layer of the ECM, which is an essential process during cell division. Here we isolated a group of actomyosin ring components that form complexes together with Chs2 at the cleavage site at the end of the cell cycle, which we named ‘ingression progression complexes’ (IPCs). In addition to type II myosin, the IQGAP protein Iqg1 and Chs2, IPCs contain the F-BAR protein Hof1, and the cytokinesis regulators Inn1 and Cyk3. We describe the molecular mechanism by which chitin synthase is activated by direct association of the C2 domain of Inn1, and the transglutaminase-like domain of Cyk3, with the catalytic domain of Chs2. We used an experimental system to find a previously unanticipated role for the C-terminus of Inn1 in preventing the untimely activation of Chs2 at the cleavage site until Cyk3 releases the block on Chs2 activity during late mitosis. These findings support a model for the co-ordinated regulation of cell division in budding yeast, in which IPCs play a central role.     

Rewiring Mid1p-Independent Medial Division in Fission Yeast

1. Download : Download high-res image (274KB)
2. Download : Download full-size image

     

Quantitative Control of Protein S-Palmitoylation Regulates Meiotic Entry in Fission Yeast

Protein S-palmitoylation, a lipid modification mediated by members of the palmitoyltransferase family, serves as an important membrane-targeting mechanism in eukaryotes. Although changes in palmitoyltransferase expression are associated with various physiological and disease states, how these changes affect global protein palmitoylation and cellular function remains unknown. Using a bioorthogonal chemical reporter and labeling strategy to identify and analyze multiple cognate substrates of a single Erf2 palmitoyltransferase, we demonstrate that control of Erf2 activity levels underlies the differential modification of key substrates such as the Rho3 GTPase in vegetative and meiotic cells. We show further that modulation of Erf2 activity levels drives changes in the palmitoylome as cells enter meiosis and affects meiotic entry. Disruption of Erf2 function delays meiotic entry, while increasing Erf2 palmitoyltransferase activity triggers aberrant meiosis in sensitized cells. Erf2-induced meiosis requires the function of the Rho3 GTPase, which is regulated by its palmitoylation state. We propose that control of palmitoyltransferase activity levels provides a fundamental mechanism for modulating palmitoylomes and cellular functions.