scholarly journals Saccharomyces cerevisiae Sps1p Regulates Trafficking of Enzymes Required for Spore Wall Synthesis

2005 ◽  
Vol 4 (3) ◽  
pp. 536-544 ◽  
Author(s):  
Michelle A. Iwamoto ◽  
Stephen R. Fairclough ◽  
Simon A. Rudge ◽  
JoAnne Engebrecht
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Specific Protein ◽  
Spore Wall ◽  
Chitin Synthase ◽  
Vegetative Cells ◽  
Glucan Synthase ◽  
Prospore Membrane ◽  
Intracellular Movement ◽  
Bud Neck ◽  
P21 Activated Kinase

ABSTRACT SPS1 encodes a sporulation-specific protein with homology to the Ste20/p21-activated kinase family. Deletion of SPS1 impinges on the formation of the spore wall, which surrounds each of the haploid nuclei generated by the meiotic divisions. Here, we demonstrate that the new internal membranes that surround the meiotic nuclei appear normal in the absence of Sps1p. Analyses of spore wall layers by immunohistochemistry suggest that the inner layers are not efficiently deposited. The defect in spore wall morphogenesis is most likely a consequence of mislocalization of enzymes required for the synthesis of the spore wall layers as both Chs3p, the major chitin synthase in yeast, and Gsc2/Fks2p, a glucan synthase transcriptionally upregulated during sporulation, fail to reach the prospore membrane in the sps1 mutant. Furthermore, localization of Chs3p to the prospore membrane is not dependent on Shc1p, a sporulation-specific homolog of Chs4p, which is required for recruitment of Chs3p to the bud neck in vegetative cells. Sps1p colocalized with Chs3p to peripheral and internal punctate structures and prospore membranes. We propose that Sps1p promotes sporulation, in part, by regulating the intracellular movement of proteins required for spore wall 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 Identification of Domains Required for Developmentally Regulated SNARE Function in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 155 (4) ◽  
pp. 1643-1655 ◽  
Author(s):  
Aaron M Neiman ◽  
Luba Katz ◽  
Patrick J Brennwald
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Snare Complex ◽  
Binding Studies ◽  
Developmentally Regulated ◽  
Vegetative Cells ◽  
Additional Layer ◽  
Prospore Membrane ◽  
N Terminus ◽  
Developmental Conditions

Abstract Saccharomyces cerevisiae cells contain two homologues of the mammalian t-SNARE protein SNAP-25, encoded by the SEC9 and SPO20 genes. Although both gene products participate in post-Golgi vesicle fusion events, they cannot substitute for one another; Sec9p is active primarily in vegetative cells while Spo20p functions only during sporulation. We have investigated the basis for the developmental stage-specific differences in the function of these two proteins. Localization of the other plasma membrane SNARE subunits, Ssop and Sncp, in sporulating cells suggests that these proteins act in conjunction with Spo20p in the formation of the prospore membrane. In vitro binding studies demonstrate that, like Sec9p, Spo20p binds specifically to the t-SNARE Sso1p and, once bound to Sso1p, can complex with the v-SNARE Snc2p. Therefore, Sec9p and Spo20p interact with the same binding partners, but developmental conditions appear to favor the assembly of complexes with Spo20p in sporulating cells. Analysis of chimeric Sec9p/Spo20p molecules indicates that regions in both the SNAP-25 domain and the unique N terminus of Spo20p are required for activity during sporulation. Additionally, the N terminus of Spo20p is inhibitory in vegetative cells. Deletion studies indicate that activation and inhibition are separable functions of the Spo20p N terminus. Our results reveal an additional layer of regulation of the SNARE complex, which is necessary only in sporulating cells.

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 Homologous Subunits of 1,3-Beta-Glucan Synthase Are Important for Spore Wall Assembly in Saccharomyces cerevisiae

2006 ◽  
Vol 6 (2) ◽  
pp. 143-156 ◽  
Author(s):  
Satoru Ishihara ◽  
Aiko Hirata ◽  
Satoru Nogami ◽  
Anne Beauvais ◽  
Jean-Paul Latge ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Elevated Temperatures ◽  
Spore Wall ◽  
Regulatory Subunit ◽  
Major Constituent ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Beta Glucan ◽  
Glucan Synthase ◽  
Wall Assembly

ABSTRACT During sporulation in Saccharomyces cerevisiae, the four haploid nuclei are encapsulated within multilayered spore walls. Glucan, the major constituent of the spore wall, is synthesized by 1,3-β-glucan synthase, which is composed of a putative catalytic subunit encoded by FKS1 and FKS2. Although another homolog, encoded by FKS3, was identified by homology searching, its function is unknown. In this report, we show that FKS2 and FKS3 are required for spore wall assembly. The ascospores of fks2 and fks3 mutants were enveloped by an abnormal spore wall with reduced resistance to diethyl ether, elevated temperatures, and ethanol. However, deletion of the FKS1 gene did not result in a defective spore wall. The construction of fusion genes that expressed Fks1p and Fks2p under the control of the FKS2 promoter revealed that asci transformed with FKS2p-driven Fks1p and Fks2p were resistant to elevated temperatures, which suggests that the expression of FKS2 plays an important role in spore wall assembly. The expression of FKS1p-driven Fks3p during vegetative growth did not affect 1,3-β-glucan synthase activity in vitro but effectively suppressed the growth defect of the temperature-sensitive fks1 mutant by stabilizing Rho1p, which is a regulatory subunit of glucan synthase. Based on these results, we propose that FKS2 encodes the primary 1,3-β-glucan synthase in sporulation and that FKS3 is required for normal spore wall formation because it affects the upstream regulation of 1,3-β-glucan synthase.

scholarly journals Specific Protein Targeting during Cell Differentiation: Polarized Localization of Fus1p during Mating Depends on Chs5p in Saccharomyces cerevisiae

2003 ◽  
Vol 2 (4) ◽  
pp. 821-825 ◽  
Author(s):  
Beatriz Santos ◽  
Michael Snyder
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Differentiation ◽  
Cell Fusion ◽  
Fusion Proteins ◽  
Protein Targeting ◽  
Specific Protein ◽  
Chitin Synthase ◽  
Chitin Synthesis ◽  
Polarized Transport ◽  
Golgi Protein

ABSTRACT In budding yeast, chs5 mutants are defective in chitin synthesis and cell fusion during mating. Chs5p is a late-Golgi protein required for the polarized transport of the chitin synthase Chs3p to the membrane. Here we show that Chs5p is also essential for the polarized targeting of Fus1p, but not of other cell fusion proteins, to the membrane during mating.

scholarly journals Sorting Signals within the Saccharomyces cerevisiae Sporulation-Specific Dityrosine Transporter, Dtr1p, C Terminus Promote Golgi-to-Prospore Membrane Transport

2008 ◽  
Vol 7 (10) ◽  
pp. 1674-1684 ◽  
Author(s):  
Masayo Morishita ◽  
JoAnne Engebrecht
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Plasma Membrane ◽  
Membrane Transport ◽  
Transmembrane Domain ◽  
Spore Wall ◽  
Bilayer Membrane ◽  
C Terminus ◽  
Prospore Membrane ◽  
Dividing Cells

ABSTRACT During sporulation in Saccharomyces cerevisiae, the dityrosine transporter Dtr1p, which is required for formation of the outermost layer of the spore wall, is specifically expressed and transported to the prospore membrane, a novel double-lipid-bilayer membrane. Dtr1p consists of 572 amino acids with predicted N- and C-terminal cytoplasmic extensions and 12 transmembrane domains. Dtr1p missing the largest internal cytoplasmic loop was trapped in the endoplasmic reticulum in both mitotically dividing cells and cells induced to sporulate. Deletion of the carboxyl 15 amino acids, but not the N-terminal extension of Dtr1p, resulted in a protein that failed to localize to the prospore membrane and was instead observed in cytoplasmic puncta. The puncta colocalized with a cis-Golgi marker, suggesting that Dtr1p missing the last 15 amino acids was trapped in an early Golgi compartment. Deletion of the C-terminal 10 amino acids resulted in a protein that localized to the prospore membrane with a delay and accumulated in cytoplasmic puncta that partially colocalized with a trans-Golgi marker. Both full-length Dtr1p and Dtr1p missing the last 10 amino acids expressed in vegetative cells localized to the plasma membrane and vacuoles, while Dtr1p deleted for the carboxyl-terminal 15 amino acids was observed only at vacuoles, suggesting that transport to the prospore membrane is mediated by distinct signals from those that specify plasma membrane localization. Transfer-of-function experiments revealed that both the carboxyl transmembrane domain and the C-terminal tail are important for Golgi complex-to-prospore membrane transport.

scholarly journals Studies on the Properties of the Sporulation Specific Protein Dit1 and Its Product Formyl Tyrosine

2020 ◽  
Vol 6 (2) ◽  
pp. 77
Author(s):  
Mostafa Basiony ◽  
Yan Yang ◽  
Guoyu Liu ◽  
Xiao-Dong Gao ◽  
Hideki Nakanishi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Specific Protein ◽  
Spore Wall ◽  
Yeast Saccharomyces Cerevisiae ◽  
Crude Cell Lysate ◽  
Unknown Mechanism ◽  
Cell Lysate ◽  
Molecule Formation ◽  
Mild Hydrolysis ◽  
Mild Acid

The dityrosine layer is a unique structure present in the spore wall of the budding yeast Saccharomyces cerevisiae. The primary constituent of this layer is bisformyl dityrosine. A sporulation-specific protein, Dit1 is localized in the spore cytosol and produces a precursor of bisformyl dityrosine. Although Dit1 is similar to isocyanide synthases, the loss of Dit1 is not rescued by heterologous expression of the Pseudomonas aeruginosa isocyanide synthase, PvcA, indicating that Dit1 does not mediate isocyanidation. The product of Dit1 is most likely formyl tyrosine. Dit1 can produce its product when it is expressed in vegetative cells; however, formyl tyrosine was not detected in the crude cell lysate. We reasoned that formyl tyrosine is unstable and reacts with some molecule to form formyl tyrosine-containing molecules in the cell lysate. In support of this hypothesis, formyl tyrosine was detected when the lysate was hydrolyzed with a mild acid. The same property was also found for bisformyl dityrosine. Bisformyl dityrosine molecules assemble to form the dityrosine layer by an unknown mechanism. Given that bisformyl dityrosine can be released from the spore wall by mild hydrolysis, the process of formyl tyrosine-containing molecule formation may resemble the assembly of the dityrosine layer.

scholarly journals The Smk1 MAPK and Its Activator, Ssp2, Are Required for Late Prospore Membrane Development in Sporulating Saccharomyces cerevisiae

2021 ◽  
Vol 7 (1) ◽  
pp. 53
Author(s):  
Matthew Durant ◽  
Joseph M. Roesner ◽  
Xheni Mucelli ◽  
Christian J. Slubowski ◽  
Erin Klee ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Map Kinase ◽  
De Novo ◽  
Leading Edge ◽  
Spore Wall ◽  
Yeast Saccharomyces Cerevisiae ◽  
Prospore Membrane ◽  
Membrane Compartments ◽  
Membrane Development

During sporulation in the budding yeast Saccharomyces cerevisiae, proper development of the prospore membrane is necessary for the formation of viable spores. The prospore membrane will eventually become the plasma membrane of the newly formed haploid spore and also serves as the template for the deposition of the spore wall. The prospore membrane is generated de novo during meiosis II and the growing edge of the prospore membrane is associated with the Leading Edge Protein (LEP) complex. We find that the Smk1 MAP kinase, along with its activator Ssp2, transiently localizes with the LEP during late meiosis II. SSP2 is required for the leading edge localization of Smk1; this localization is independent of the activation state of Smk1. Like other LEP components, the localization of Smk1 at the leading edge also depends on Ady3. Although prospore membrane development begins normally in smk1 and ssp2 mutants, late prospore membrane formation is disrupted, with the formation of ectopic membrane compartments. Thus, MAP kinase signaling plays an important role in the formation of the prospore membrane.

scholarly journals Dtr1p, a Multidrug Resistance Transporter of the Major Facilitator Superfamily, Plays an Essential Role in Spore Wall Maturation in Saccharomyces cerevisiae

2002 ◽  
Vol 1 (5) ◽  
pp. 799-810 ◽  
Author(s):  
Thomas Felder ◽  
Edith Bogengruber ◽  
Sandra Tenreiro ◽  
Adi Ellinger ◽  
Isabel Sá-Correia ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Multidrug Resistance ◽  
De Novo ◽  
Physiological Role ◽  
Spore Wall ◽  
Major Facilitator Superfamily ◽  
Spore Surface ◽  
Prospore Membrane ◽  
A Genome ◽  
Major Facilitator

ABSTRACT The de novo formation of multilayered spore walls inside a diploid mother cell is a major landmark of sporulation in the yeast Saccharomyces cerevisiae. Synthesis of the dityrosine-rich outer spore wall takes place toward the end of this process. Bisformyl dityrosine, the major building block of the spore surface, is synthesized in a multistep process in the cytoplasm of the prospores, transported to the maturing wall, and polymerized into a highly cross-linked macromolecule on the spore surface. Here we present evidence that the sporulation-specific protein Dtr1p (encoded by YBR180w) plays an important role in spore wall synthesis by facilitating the translocation of bisformyl dityrosine through the prospore membrane. DTR1 was identified in a genome-wide screen for spore wall mutants. The null mutant accumulates unusually large amounts of bisformyl dityrosine in the cytoplasm and fails to efficiently incorporate this precursor into the spore surface. As a result, many mutant spores have aberrant surface structures. Dtr1p, a member of the poorly characterized DHA12 (drug:H+ antiporter with 12 predicted membrane spans) family, is localized in the prospore membrane throughout spore maturation. Transport by Dtr1p may not be restricted to its natural substrate, bisformyl dityrosine. When expressed in vegetative cells, Dtr1p renders these cells slightly more resistant against unrelated toxic compounds, such as antimalarial drugs and food-grade organic acid preservatives. Dtr1p is the first multidrug resistance protein of the major facilitator superfamily with an assigned physiological role in the yeast cell.

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