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.

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 The Dysferlin Domain-Only Protein, Spo73, Is Required for Prospore Membrane Extension in Saccharomyces cerevisiae

mSphere ◽  
2015 ◽  
Vol 1 (1) ◽  
Author(s):  
Yuuya Okumura ◽  
Tsuyoshi S. Nakamura ◽  
Takayuki Tanaka ◽  
Ichiro Inoue ◽  
Yasuyuki Suda ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Developmental Process ◽  
Spore Wall ◽  
Membrane Formation ◽  
Content Type ◽  
Prospore Membrane ◽  
Proper Size ◽  
Membrane Extension ◽  
Extension Analysis

ABSTRACT Prospore membrane formation consists of de novo double-membrane formation, which occurs during the developmental process of sporulation in Saccharomyces cerevisiae. Membranes are formed into their proper size and shape, and thus, prospore membrane formation has been studied as a general model of membrane formation. We identified SPO73, previously shown to be required for spore wall formation, as an additional gene involved in prospore membrane extension. Genetic and cell biological analyses suggested that Spo73 functions on the prospore membrane with other factors in prospore membrane extension, counteracting the bending force of the prospore membrane. Spo73 is the first dysferlin domain-only protein ever analyzed. The dysferlin domain is conserved from yeast to mammals and is found in dysferlin proteins, which are involved in dysferlinopathy, although the precise function of the domain is unknown. Continued analysis of Spo73 will contribute to our understanding of the function of dysferlin domains and dysferlinopathy. Sporulation of Saccharomyces cerevisiae is a developmental process in which an ascus containing four haploid spores forms from a diploid cell. During this process, newly formed membrane structures called prospore membranes extend along the nuclear envelope and engulf and package daughter nuclei along with cytosol and organelles to form precursors of spores. Proteins involved in prospore membrane extension, Vps13 and Spo71, have recently been reported; however, the overall mechanism of membrane extension remains unclear. Here, we identified Spo73 as an additional factor involved in prospore membrane extension. Analysis of a spo73∆ mutant revealed that it shows defects similar to those of a spo71∆ mutant during prospore membrane formation. Spo73 localizes to the prospore membrane, and this localization is independent of Spo71 and Vps13. In contrast, a Spo73 protein carrying mutations in a surface basic patch mislocalizes to the cytoplasm and overexpression of Spo71 can partially rescue localization to the prospore membrane. Similar to spo71∆ mutants, spo73∆ mutants display genetic interactions with the mutations in the SMA2 and SPO1 genes involved in prospore membrane bending. Further, our bioinformatic analysis revealed that Spo73 is a dysferlin domain-only protein. Thus, these results suggest that a dysferlin domain-only protein, Spo73, functions with a dual pleckstrin homology domain protein, Spo71, in prospore membrane extension. Analysis of Spo73 will provide insights into the conserved function of dysferlin domains, which is related to dysferlinopathy. IMPORTANCE Prospore membrane formation consists of de novo double-membrane formation, which occurs during the developmental process of sporulation in Saccharomyces cerevisiae. Membranes are formed into their proper size and shape, and thus, prospore membrane formation has been studied as a general model of membrane formation. We identified SPO73, previously shown to be required for spore wall formation, as an additional gene involved in prospore membrane extension. Genetic and cell biological analyses suggested that Spo73 functions on the prospore membrane with other factors in prospore membrane extension, counteracting the bending force of the prospore membrane. Spo73 is the first dysferlin domain-only protein ever analyzed. The dysferlin domain is conserved from yeast to mammals and is found in dysferlin proteins, which are involved in dysferlinopathy, although the precise function of the domain is unknown. Continued analysis of Spo73 will contribute to our understanding of the function of dysferlin domains and dysferlinopathy.

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.

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.

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 Nsk1 ensures accurate chromosome segregation by promoting association of kinetochores to spindle poles during anaphase B

2011 ◽  
Vol 22 (23) ◽  
pp. 4486-4502 ◽  
Author(s):  
Graham J. Buttrick ◽  
John C. Meadows ◽  
Theresa C. Lancaster ◽  
Vincent Vanoosthuyse ◽  
Lindsey A. Shepperd ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Growth Defect ◽  
Catalytic Subunits ◽  
Spindle Poles ◽  
Sister Chromatids ◽  
Anaphase B ◽  
Novel Protein

Type 1 phosphatase (PP1) antagonizes Aurora B kinase to stabilize kinetochore–microtubule attachments and to silence the spindle checkpoint. We screened for factors that exacerbate the growth defect of Δdis2 cells, which lack one of two catalytic subunits of PP1 in fission yeast, and identified Nsk1, a novel protein required for accurate chromosome segregation. During interphase, Nsk1 resides in the nucleolus but spreads throughout the nucleoplasm as cells enter mitosis. Following dephosphorylation by Clp1 (Cdc14-like) phosphatase and at least one other phosphatase, Nsk1 localizes to the interface between kinetochores and the inner face of the spindle pole body during anaphase. In the absence of Nsk1, some kinetochores become detached from spindle poles during anaphase B. If this occurs late in anaphase B, then the sister chromatids of unclustered kinetochores segregate to the correct daughter cell. These unclustered kinetochores are efficiently captured, retrieved, bioriented, and segregated during the following mitosis, as long as Dis2 is present. However, if kinetochores are detached from a spindle pole early in anaphase B, then these sister chromatids become missegregated. These data suggest Nsk1 ensures accurate chromosome segregation by promoting the tethering of kinetochores to spindle poles during anaphase B.

scholarly journals Novelsfi1Alleles Uncover Additional Functions for Sfi1p in Bipolar Spindle Assembly and Function

2007 ◽  
Vol 18 (6) ◽  
pp. 2047-2056 ◽  
Author(s):  
Victoria E. Anderson ◽  
John Prudden ◽  
Simon Prochnik ◽  
Thomas H. Giddings ◽  
Kevin G. Hardwick
Keyword(s):  
Essential Gene ◽  
Light And Electron Microscopy ◽  
Spindle Pole Body ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
The Novel ◽  
Synthetic Lethal ◽  
Bipolar Spindle ◽  
Novel Alleles ◽  
And Function

A variety of spindle and kinetochore defects have been shown to induce a mitotic delay through activation of the spindle checkpoint. With the aim of identifying novel mitotic defects we carried out a mad1 synthetic lethal screen in budding yeast. In this screen, four novel alleles of sfi1 were isolated. SFI1 is an essential gene, previously identified through its interaction with centrin/CDC31 and shown to be required for spindle pole body (SPB) duplication. The new mutations were all found in the C-terminal domain of Sfi1p, which has no known function, but it is well conserved among budding yeasts. Analysis of the novel sfi1 mutants, through a combination of light and electron microscopy, revealed duplicated SPBs <0.3 μm apart. Importantly, these SPBs have completed duplication, but they are not separated, suggesting a possible defect in splitting of the bridge. We discuss possible roles for Sfi1p in this step in bipolar spindle assembly.

scholarly journals Role of Septins in the Orientation of Forespore Membrane Extension during Sporulation in Fission Yeast

2010 ◽  
Vol 30 (8) ◽  
pp. 2057-2074 ◽  
Author(s):  
Masayuki Onishi ◽  
Takako Koga ◽  
Aiko Hirata ◽  
Taro Nakamura ◽  
Haruhiko Asakawa ◽  
...  
Keyword(s):  
Spindle Pole Body ◽  
Leading Edge ◽  
Spindle Pole ◽  
Pole Body ◽  
Membrane Extension ◽  
Fusion Site ◽  
Phosphatidylinositol 4 ◽  
Ring Shaped Structure

ABSTRACT During yeast sporulation, a forespore membrane (FSM) initiates at each spindle-pole body and extends to form the spore envelope. We used Schizosaccharomyces pombe to investigate the role of septins during this process. During the prior conjugation of haploid cells, the four vegetatively expressed septins (Spn1, Spn2, Spn3, and Spn4) coassemble at the fusion site and are necessary for its normal morphogenesis. Sporulation involves a different set of four septins (Spn2, Spn5, Spn6, and the atypical Spn7) that does not include the core subunits of the vegetative septin complex. The four sporulation septins form a complex in vitro and colocalize interdependently to a ring-shaped structure along each FSM, and septin mutations result in disoriented FSM extension. The septins and the leading-edge proteins appear to function in parallel to orient FSM extension. Spn2 and Spn7 bind to phosphatidylinositol 4-phosphate [PtdIns(4)P] in vitro, and PtdIns(4)P is enriched in the FSMs, suggesting that septins bind to the FSMs via this lipid. Cells expressing a mutant Spn2 protein unable to bind PtdIns(4)P still form extended septin structures, but these structures fail to associate with the FSMs, which are frequently disoriented. Thus, septins appear to form a scaffold that helps to guide the oriented extension of the FSM.

scholarly journals A Fourth Component of the Fission Yeast γ-Tubulin Complex, Alp16, Is Required for Cytoplasmic Microtubule Integrity and Becomes Indispensable When γ-Tubulin Function Is Compromised

2002 ◽  
Vol 13 (7) ◽  
pp. 2360-2373 ◽  
Author(s):  
Akiko Fujita ◽  
Leah Vardy ◽  
Miguel Angel Garcia ◽  
Takashi Toda
Keyword(s):  
Fission Yeast ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Microtubule Organizing Center ◽  
Cytoplasmic Microtubule ◽  
Multicopy Suppressor ◽  
Large Complex ◽  
Synthetically Lethal ◽  
And Function

γ-Tubulin functions as a multiprotein complex, called the γ-tubulin complex (γ-TuC), and composes the microtubule organizing center (MTOC). Fission yeast Alp4 and Alp6 are homologues of two conserved γ-TuC proteins, hGCP2 and hGCP3, respectively. We isolated a novel gene, alp16 + , as a multicopy suppressor of temperature-sensitive alp6-719mutants. alp16 + encodes a 759-amino-acid protein with two conserved regions found in all other members of γ-TuC components. In addition, Alp16 contains an additional motif, which shows homology to hGCP6/Xgrip210. Gene disruption shows that alp16 + is not essential for cell viability. However, alp16 deletion displays abnormally long cytoplasmic microtubules, which curve around the cell tip. Furthermore, alp16-deleted mutants are hypersensitive to microtubule-depolymerizing drugs and synthetically lethal with either temperature-sensitive alp4-225,alp4-1891, or alp6-719 mutants. Overproduction of Alp16 is lethal, with defective phenotypes very similar to loss of Alp4 or Alp6. Alp16 localizes to the spindle pole body throughout the cell cycle and to the equatorial MTOC at postanaphase. Alp16 coimmunoprecipitates with γ-tubulin and cosediments with the γ-TuC in a large complex (>20 S). Alp16 is, however, not required for the formation of this large complex. We discuss evolutional conservation and divergence of structure and function of the γ-TuC between yeast and higher eukaryotes.

scholarly journals Vesicle Docking to the Spindle Pole Body Is Necessary to Recruit the Exocyst During Membrane Formation inSaccharomyces cerevisiae

2010 ◽  
Vol 21 (21) ◽  
pp. 3693-3707 ◽  
Author(s):  
Erin M. Mathieson ◽  
Yasuyuki Suda ◽  
Mark Nickas ◽  
Brian Snydsman ◽  
Trisha N. Davis ◽  
...  
Keyword(s):  
De Novo ◽  
Spindle Pole Body ◽  
Coiled Coil ◽  
Resonance Energy ◽  
Initiation Site ◽  
Spindle Pole ◽  
Rab Gtpase ◽  
Membrane Formation ◽  
Vesicle Docking ◽  
Pole Body

During meiosis II in Saccharomyces cerevisiae, the cytoplasmic face of the spindle pole body, referred to as the meiosis II outer plaque (MOP), is modified in both composition and structure to become the initiation site for de novo formation of a membrane called the prospore membrane. The MOP serves as a docking complex for precursor vesicles that are targeted to its surface. Using fluorescence resonance energy transfer analysis, the orientation of coiled-coil proteins within the MOP has been determined. The N-termini of two proteins, Mpc54p and Spo21p, were oriented toward the outer surface of the structure. Mutations in the N-terminus of Mpc54p resulted in a unique phenotype: precursor vesicles loosely tethered to the MOP but did not contact its surface. Thus, these mpc54 mutants separate the steps of vesicle association and docking. Using these mpc54 mutants, we determined that recruitment of the Rab GTPase Sec4p, as well as the exocyst components Sec3p and Sec8p, to the precursor vesicles requires vesicle docking to the MOP. This suggests that the MOP promotes membrane formation both by localization of precursor vesicles to a particular site and by recruitment of a second tethering complex, the exocyst, that stimulates downstream events of fusion.

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