scholarly journals Phosphatidylinositol-4,5-Bisphosphate and Phospholipase D-Generated Phosphatidic Acid Specify SNARE-Mediated Vesicle Fusion for Prospore Membrane Formation

2009 ◽  
Vol 8 (8) ◽  
pp. 1094-1105 ◽  
Author(s):  
Rima Mendonsa ◽  
JoAnne Engebrecht
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Fluorescent Protein ◽  
De Novo ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Vesicle Fusion ◽  
Protein Receptor ◽  
Prospore Membrane

ABSTRACT The soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) family of proteins is required for eukaryotic intracellular membrane fusions. Vesicle fusion for formation of the prospore membrane (PSM), a membrane compartment that forms de novo during yeast sporulation, requires SNARE function, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], and the activity of the phospholipase D (PLD) Spo14p, which generates phosphatidic acid (PA). The SNARE syntaxin Sso1p is essential for PSM production while the functionally redundant homolog in vegetative growth, Sso2p, is not. We demonstrate that Sso1p and Sso2p bind similarly in vitro to PA or phosphoinositide-containing liposomes and that the conserved SNARE (H3) domain largely mediates PA-binding. Both green fluorescent protein-Sso fusion proteins localize to the developing PSM in wild-type cells and to the spindle pole body in spo14Δ cells induced to sporulate. However, the autoregulatory region of Sso1p binds PI(4,5)P2-containing liposomes in vitro with a greater ability than the equivalent region of Sso2p. Overexpression of the phosphatidylinositol-4-phosphate 5-kinase MSS4 in sso1Δ cells induced to sporulate stimulates PSM production; PLD activity is not increased under these conditions, indicating that PI(4,5)P2 has roles in addition to stimulating PLD in PSM formation. These data suggest that PLD-generated PA and PI(4,5)P2 collaborate at multiple levels to promote SNARE-mediated fusion for PSM formation.

Ady3p Links Spindle Pole Body Function to Spore Wall Synthesis in Saccharomyces cerevisiae

Genetics ◽  
2002 ◽  
Vol 160 (4) ◽  
pp. 1439-1450
Author(s):  
Mark E Nickas ◽  
Aaron M Neiman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Spindle Pole Body ◽  
Leading Edge ◽  
Spindle Pole ◽  
Spore Wall ◽  
Pole Body ◽  
Prospore Membrane ◽  
Sporulation Efficiency ◽  
Spore Walls

Abstract Spore formation in Saccharomyces cerevisiae requires the de novo synthesis of prospore membranes and spore walls. Ady3p has been identified as an interaction partner for Mpc70p/Spo21p, a meiosis-specific component of the outer plaque of the spindle pole body (SPB) that is required for prospore membrane formation, and for Don1p, which forms a ring-like structure at the leading edge of the prospore membrane during meiosis II. ADY3 expression has been shown to be induced in midsporulation. We report here that Ady3p interacts with additional components of the outer and central plaques of the SPB in the two-hybrid assay. Cells that lack ADY3 display a decrease in sporulation efficiency, and most ady3Δ/ady3Δ asci that do form contain fewer than four spores. The sporulation defect in ady3Δ/ady3Δ cells is due to a failure to synthesize spore wall polymers. Ady3p forms ring-like structures around meiosis II spindles that colocalize with those formed by Don1p, and Don1p rings are absent during meiosis II in ady3Δ/ady3Δ cells. In mpc70Δ/mpc70Δ cells, Ady3p remains associated with SPBs during meiosis II. Our results suggest that Ady3p mediates assembly of the Don1p-containing structure at the leading edge of the prospore membrane via interaction with components of the SPB and that this structure is involved in spore wall formation.

scholarly journals Dynamic localization of a yeast development–specific PP1 complex during prospore membrane formation is dependent on multiple localization signals and complex formation

2017 ◽  
Vol 28 (26) ◽  
pp. 3881-3895 ◽  
Author(s):  
Tsuyoshi S. Nakamura ◽  
Yumi Numajiri ◽  
Yuuya Okumura ◽  
Junji Hidaka ◽  
Takayuki Tanaka ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
De Novo ◽  
Developmental Process ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Membrane Structures ◽  
Prospore Membrane ◽  
Membrane Extension ◽  
And Function

During the developmental process of sporulation in Saccharomyces cerevisiae, membrane structures called prospore membranes are formed de novo, expand, extend, acquire a round shape, and finally become plasma membranes of the spores. GIP1 encodes a regulatory/targeting subunit of protein phosphatase type 1 that is required for sporulation. Gip1 recruits the catalytic subunit Glc7 to septin structures that form along the prospore membrane; however, the molecular basis of its localization and function is not fully understood. Here we show that Gip1 changes its localization dynamically and is required for prospore membrane extension. Gip1 first associates with the spindle pole body as the prospore membrane forms, moves onto the prospore membrane and then to the septins as the membrane extends, distributes around the prospore membrane after closure, and finally translocates into the nucleus in the maturing spore. Deletion and mutation analyses reveal distinct sequences in Gip1 that are required for different localizations and for association with Glc7. Binding to Glc7 is also required for proper localization. Strikingly, localization to the prospore membrane, but not association with septins, is important for Gip1 function. Further, our genetic analysis suggests that a Gip1–Glc7 phosphatase complex regulates prospore membrane extension in parallel to the previously reported Vps13, Spo71, Spo73 pathway.

scholarly journals Positive and Negative Regulation of a SNARE Protein by Control of Intracellular Localization

2004 ◽  
Vol 15 (4) ◽  
pp. 1802-1815 ◽  
Author(s):  
Hideki Nakanishi ◽  
Pablo de los Santos ◽  
Aaron M. Neiman
Keyword(s):  
Fluorescent Protein ◽  
Genetic Manipulation ◽  
Intracellular Localization ◽  
Negative Regulation ◽  
Snare Protein ◽  
Nuclear Targeting ◽  
Protein Receptor ◽  
Prospore Membrane

In Saccharomyces cerevisiae, the developmentally regulated Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein Spo20p mediates the fusion of vesicles with the prospore membrane, which is required for the formation of spores. Spo20p is subject to both positive and negative regulation by separate sequences in its aminoterminal domain. We report that the positive activity is conferred by a short, amphipathic helix that is sufficient to confer plasma membrane or prospore membrane localization to green fluorescent protein. In vitro, this helix binds to acidic phospholipids, and mutations that reduce or eliminate phospholipid binding in vitro inactivate Spo20p in vivo. Genetic manipulation of phospholipid pools indicates that the likely in vivo ligand of this domain is phosphatidic acid. The inhibitory activity is a nuclear targeting signal, which confers nuclear localization in vegetative cells and in cells entering meiosis. However, as cells initiate spore formation, fusions containing the inhibitory domain exit the nucleus and localize to the nascent prospore membrane. Thus, the SNARE Spo20p is both positively and negatively regulated by control of its intracellular localization.

scholarly journals Ady4p and Spo74p Are Components of the Meiotic Spindle Pole Body That Promote Growth of the Prospore Membrane in Saccharomyces cerevisiae

2003 ◽  
Vol 2 (3) ◽  
pp. 431-445 ◽  
Author(s):  
Mark E. Nickas ◽  
Cindi Schwartz ◽  
Aaron M. Neiman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Meiotic Spindle ◽  
Membrane Formation ◽  
Auxiliary Component ◽  
Pole Body ◽  
Prospore Membrane ◽  
Organizing Center

ABSTRACT Spore formation in Saccharomyces cerevisiae occurs via the de novo synthesis of the prospore membrane during the second meiotic division. Prospore membrane formation is triggered by assembly of a membrane-organizing center, the meiotic outer plaque (MOP), on the cytoplasmic face of the spindle pole body (SPB) during meiosis. We report here the identification of two new components of the MOP, Ady4p and Spo74p. Ady4p and Spo74p interact with known proteins of the MOP and are localized to the outer plaque of the SPB during meiosis II. MOP assembly and prospore membrane formation are abolished in spo74Δ/spo74Δ cells and occur aberrantly in ady4Δ/ady4Δ cells. Spo74p and the MOP component Mpc70p are mutually dependent for recruitment to SPBs during meiosis. In contrast, both Ady4p and Spo74p are present at SPBs, albeit at reduced levels, in cells that lack the MOP component Mpc54p. Our findings suggest a model for the assembled MOP in which Mpc54p, Mpc70p, and Spo74p make up a core structural unit of the scaffold that initiates synthesis of the prospore membrane, and Ady4p is an auxiliary component that stabilizes the plaque.

Phospholipase D1b and D2a generate structurally identical phosphatidic acid species in mammalian cells

2001 ◽  
Vol 360 (3) ◽  
pp. 707-715 ◽  
Author(s):  
Trevor R. PETTITT ◽  
Mark McDERMOTT ◽  
Khalid M. SAQIB ◽  
Neil SHIMWELL ◽  
Michael J. O. WAKELAM
Keyword(s):  
Liquid Chromatography ◽  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Mammalian Cells ◽  
Inducible Promoter ◽  
In Vitro Assays ◽  
Protein Levels ◽  
Intracellular Messenger

Mammalian cells contain different phospholipase D enzymes (PLDs) whose distinct physiological roles are poorly understood and whose products have not been characterized. The development of porcine aortic endothelial (PAE) cell lines able to overexpress PLD-1b or −2a under the control of an inducible promoter has enabled us to characterize both the substrate specificity and the phosphatidic acid (PtdOH) product of these enzymes under controlled conditions. Liquid chromatography–MS analysis showed that PLD1b- and PLD2a-transfected PAE cells, as well as COS7 and Rat1 cells, generate similar PtdOH and, in the presence of butan-1-ol, phosphatidylbutanol (PtdBut) profiles, enriched in mono- and di-unsaturated species, in particular 16:0/18:1. Although PtdBut mass increased, the species profile did not change in cells stimulated with ATP or PMA. Overexpression of PLD made little difference to basal or stimulated PtdBut formation, indicating that activity is tightly regulated in vivo and that factors other than just PLD protein levels limit hydrolytic function. In vitro assays using PLD-enriched lysates showed that the enzyme could utilize both phosphatidylcholine and, much less efficiently, phosphatidylethanolamine, with slight selectivity towards mono- and di-unsaturated species. Phosphatidylinositol was not a substrate. Thus PLD1b and PLD2a hydrolyse a structurally similar substrate pool to generate an identical PtdOH product enriched in mono- and di-unsaturated species that we propose to function as the intracellular messenger forms of this lipid.

Cytoplasmic microtubular system implicated in de novo formation of a Rabl-like orientation of chromosomes in fission yeast

2001 ◽  
Vol 114 (13) ◽  
pp. 2427-2435 ◽  
Author(s):  
Bunshiro Goto ◽  
Koei Okazaki ◽  
Osami Niwa
Keyword(s):  
Fission Yeast ◽  
De Novo ◽  
Nuclear Periphery ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Limited Area ◽  
Cytoplasmic Microtubule ◽  
Random Arrangement ◽  
Triplex Structure ◽  
Microtubular System

Chromosomes are not packed randomly in the nucleus. The Rabl orientation is an example of the non-random arrangement of chromosomes, centromeres are grouped in a limited area near the nuclear periphery and telomeres are located apart from centromeres. This orientation is established during mitosis and maintained through subsequent interphase in a range of species. We report that a Rabl-like configuration can be formed de novo without a preceding mitosis during the transition from the sexual phase to the vegetative phase of the life cycle in fission yeast. In this process, each of the dispersed centromeres is often associated with a novel Sad1-containing body that is contacting a cytoplasmic microtubule laterally (Sad1 is a component of the spindle pole body (SPB)). The Sad1-containing body was colocalized with other known SPB components, Kms1 and Spo15 but not with Cut12, indicating that it represents a novel SPB-related complex. The existence of the triplex structure (centromere-microtubule-Sad1 body) suggests that the clustering of centromeres is controlled by a cytoplasmic microtubular system. Accordingly, when microtubules are destabilized, clustering is markedly reduced.

scholarly journals Helminth secretions induce de novo T cell Foxp3 expression and regulatory function through the TGF-β pathway

2010 ◽  
Vol 207 (11) ◽  
pp. 2331-2341 ◽  
Author(s):  
John R. Grainger ◽  
Katie A. Smith ◽  
James P. Hewitson ◽  
Henry J. McSorley ◽  
Yvonne Harcus ◽  
...  
Keyword(s):  
Transforming Growth Factor ◽  
Fluorescent Protein ◽  
De Novo ◽  
Foxp3 Expression ◽  
Dominant Negative ◽  
Regulatory Function ◽  
Intestinal Helminth ◽  
Helminth Parasites

Foxp3-expressing regulatory T (T reg) cells have been implicated in parasite-driven inhibition of host immunity during chronic infection. We addressed whether parasites can directly induce T reg cells. Foxp3 expression was stimulated in naive Foxp3− T cells in mice infected with the intestinal helminth Heligmosomoides polygyrus. In vitro, parasite-secreted proteins (termed H. polygyrus excretory-secretory antigen [HES]) induced de novo Foxp3 expression in fluorescence-sorted Foxp3− splenocytes from Foxp3–green fluorescent protein reporter mice. HES-induced T reg cells suppressed both in vitro effector cell proliferation and in vivo allergic airway inflammation. HES ligated the transforming growth factor (TGF) β receptor and promoted Smad2/3 phosphorylation. Foxp3 induction by HES was lost in dominant-negative TGF-βRII cells and was abolished by the TGF-β signaling inhibitor SB431542. This inhibitor also reduced worm burdens in H. polygyrus–infected mice. HES induced IL-17 in the presence of IL-6 but did not promote Th1 or Th2 development under any conditions. Importantly, antibody to mammalian TGF-β did not recognize HES, whereas antisera that inhibited HES did not affect TGF-β. Foxp3 was also induced by secreted products of Teladorsagia circumcincta, a related nematode which is widespread in ruminant animals. We have therefore identified a novel pathway through which helminth parasites may stimulate T reg cells, which is likely to be a key part of the parasite’s immunological relationship with the host.

scholarly journals Stu2p: A Microtubule-Binding Protein that Is an Essential Component of the Yeast Spindle Pole Body

1997 ◽  
Vol 139 (5) ◽  
pp. 1271-1280 ◽  
Author(s):  
Peijing Jeremy Wang ◽  
Tim C. Huffaker
Keyword(s):  
Essential Gene ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Cold Sensitivity ◽  
Gene Encoding ◽  
Microtubule Binding ◽  
Amino Acid Region ◽  
Pole Body ◽  
Spindle Function

Previously we isolated tub2-423, a cold-sensitive allele of the Saccharomyces cerevisiae gene encoding β-tubulin that confers a defect in mitotic spindle function. In an attempt to identify additional proteins that are important for spindle function, we screened for suppressors of the cold sensitivity of tub2-423 and obtained two alleles of a novel gene, STU2. STU2 is an essential gene and encodes a protein whose sequence is similar to proteins identified in a variety of organisms. Stu2p localizes primarily to the spindle pole body (SPB) and to a lesser extent along spindle microtubules. Localization to the SPB is not dependent on the presence of microtubules, indicating that Stu2p is an integral component of the SPB. Stu2p also binds microtubules in vitro. We have localized the microtubule-binding domain of Stu2p to a highly basic 100-amino acid region. This region contains two imperfect repeats; both repeats appear to contribute to microtubule binding to similar extents. These results suggest that Stu2p may play a role in the attachment, organization, and/or dynamics of microtubule ends at the SPB.

Phospholipase D: enzymology, mechanisms of regulation, and function

1997 ◽  
Vol 77 (2) ◽  
pp. 303-320 ◽  
Author(s):  
J. H. Exton
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phosphatidic Acid ◽  
G Protein ◽  
Phospholipase D ◽  
Tyrosine Kinases ◽  
Rho Proteins ◽  
Kinase C

Phospholipase D exists in various forms that differ in their regulation but predominantly hydrolyze phosphatidylcholine. The Ca(2+)-dependent isozymes of protein kinase C regulate phospholipase D in vitro and play a major role in its control by growth factors and G protein-linked agonists in vivo. Recent studies have demonstrated that small G proteins of the ADP-ribosylation factor (ARF) and Rho families activate the enzyme in vitro, and evidence is accumulating that they also are involved in its control in vivo. Both types of G protein play important roles in cellular function, and the possible mechanisms by which they are activated by agonists are discussed. There is also emerging evidence of the control of phospholipase D and Rho proteins by soluble tyrosine kinases and novel serine/threonine kinases. The possible role of these kinases in agonist regulation of phospholipase D is discussed. The function of phospholipase D in cells is still poorly defined. Postulated roles of phosphatidic acid produced by phospholipase D action include the activation of Ca(2+)-independent isoforms of protein kinase C, the regulation of growth and the cytoskeleton in fibroblasts, and control of the respiratory burst in neutrophils. Another important function of phosphatidic acid is to act as a substrate for a specific phospholipase A2 to generate lysophosphatidic acid, which is becoming increasingly recognized as a major intercellular messenger. Finally, it is possible that the phospholipid changes induced in various cellular membranes by phospholipase D may per se play an important role in vesicle trafficking and other membrane-associated events.

scholarly journals Identification of Ribonucleotide Reductase Protein R1 as an Activator of Microtubule Nucleation in Xenopus Egg Mitotic Extracts

2000 ◽  
Vol 11 (12) ◽  
pp. 4173-4187 ◽  
Author(s):  
Saeko Takada ◽  
Takehiko Shibata ◽  
Yasushi Hiraoka ◽  
Hirohisa Masuda
Keyword(s):  
Ribonucleotide Reductase ◽  
Large Subunit ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Microtubule Nucleation ◽  
Pole Body ◽  
Recombinant Mouse ◽  
Xenopus Egg ◽  
Essential Subunit

Microtubule nucleation on the centrosome and the fungal equivalent, the spindle pole body (SPB), is activated at the onset of mitosis. We previously reported that mitotic extracts prepared fromXenopus unfertilized eggs convert the interphase SPB of fission yeast into a competent state for microtubule nucleation. In this study, we have purified an 85-kDa SPB activator from the extracts and identified it as the ribonucleotide reductase large subunit R1. We further confirmed that recombinant mouse R1 protein was also effective for SPB activation. On the other hand, another essential subunit of ribonucleotide reductase, R2 protein, was not required for SPB activation. SPB activation by R1 protein was suppressed in the presence of anti-R1 antibodies or a partial oligopeptide of R1; the oligopeptide also inhibited aster formation on Xenopussperm centrosomes. In accordance, R1 was detected in animal centrosomes by immunofluorescence and immunoblotting with anti-R1 antibodies. In addition, recombinant mouse R1 protein bound to γ- and α/β-tubulin in vitro. These results suggest that R1 is a bifunctional protein that acts on both ribonucleotide reduction and centrosome/SPB activation.

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