In vitro reactions of vacuole inheritance in Saccharomyces cerevisiae.

Vacuole inheritance is temporally coordinated with the cell cycle and is restricted spatially to an axis between the maternal vacuole and the bud. The new bud vacuole is founded by a stream of vacuole-derived membranous vesicles and tubules which are transported from the mother cell into the bud to form the daughter organelle. We now report in vitro formation of vacuole-derived tubules and vesicles. In semi-intact cells, formation of tubulovesicular structures requires ATP and the proteins encoded by VAC1 and VAC2, two genes which are required for vacuole inheritance in vivo. Isolation of vacuoles from cell lysates before in vitro incubation reveals that formation of tubulovesicular structures requires cytosol as well as ATP. After forming tubulovesicular structures, isolated vacuoles subsequently increase in size. Biochemical assays reveal that this increase results from vacuole to vacuole fusion, leading to mixing of organellar contents. Intervacuolar fusion is sensitive to the phosphatase inhibitors microcystin-LR and okadaic acid, suggesting that protein phosphorylation/dephosphorylation reactions play a role in this event.

Download Full-text

Thioredoxin is required for vacuole inheritance in Saccharomyces cerevisiae.

The Journal of Cell Biology ◽

10.1083/jcb.132.5.787 ◽

1996 ◽

Vol 132 (5) ◽

pp. 787-794 ◽

Cited By ~ 56

Author(s):

Z Xu ◽

W Wickner

Keyword(s):

Saccharomyces Cerevisiae ◽

Molecular Weight ◽

Fusion Reaction ◽

S Phase ◽

Low Molecular Weight ◽

Vacuole Fusion ◽

Vacuole Inheritance ◽

Segregation Structure

The vacuole of Saccharomyces cerevisiae projects a stream of tubules a and vesicles (a "segregation structure") into the bud in early S phase. We have described an in vitro reaction, requiring physiological temperature, ATP, and cytosol, in which isolated vacuoles form segregation structures and fuse. This in vitro reaction is defective when reaction components are prepared from vac mutants that are defective in this process in vivo, Fractionation of the cytosol reveals at least three components, each of which can support the vacuole fusion reaction, and two stimulatory fractions. Purification of one "low molecular weight activity" (LMA1) yields a heterodimeric protein with a thioredoxin subunit. Most of the thioredoxin of yeast is in this complex rather than the well-studied monomer. A deletion of both S. cerevisiae thioredoxin genes causes a striking vacuole inheritance defect in vivo, establishing a role for thioredoxin as a novel factor in this trafficking reaction.

Download Full-text

G-protein ligands inhibit in vitro reactions of vacuole inheritance.

The Journal of Cell Biology ◽

10.1083/jcb.126.1.87 ◽

1994 ◽

Vol 126 (1) ◽

pp. 87-97 ◽

Cited By ~ 125

Author(s):

A Haas ◽

B Conradt ◽

W Wickner

Keyword(s):

Alkaline Phosphatase ◽

Vacuole Membrane ◽

Protein Ligands ◽

Vacuole Fusion ◽

Proteinase A ◽

Membrane Bound ◽

Vacuole Inheritance ◽

Cytosolic Factors

During budding in Saccharomyces cerevisiae, maternal vacuole material is delivered into the growing daughter cell via tubular or vesicular structures. One of the late steps in vacuole inheritance is the fusion in the bud of vesicles derived from the maternal vacuole. This process has been reconstituted in vitro and requires isolated vacuoles, a physiological temperature, cytosolic factors, and ATP (Conradt, B., J. Shaw, T. Vida, S. Emr, and W. Wickner. 1992. J. Cell Biol. 119:1469-1479). We now report a simple and reliable assay to quantify vacuole-to-vacuole fusion in vitro. This assay is based on the maturation and activation of vacuole membrane-bound pro-alkaline phosphatase by vacuolar proteinase A after vacuole-to-vacuole fusion. In vitro fusion allowed maturation of 30 to 60% of pro-alkaline phosphatase. Vacuoles prepared from a mutant defective in vacuole inheritance in vivo (vac2-1) were inactive in this assay. Vacuole fusion in vitro required a vacuole membrane potential. Inhibition by nonhydrolyzable guanosine derivatives, mastoparans, and benzalkonium chloride suggest that GTP-hydrolyzing G proteins may play a key role in the in vitro fusion events.

Download Full-text

A RAD9-dependent cell cycle arrest in response to unresolved recombination intermediates in Saccharomyces cerevisiae

10.1101/736850 ◽

2019 ◽

Author(s):

Hardeep Kaur ◽

GN Krishnaprasad ◽

Michael Lichten

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Dna Damage ◽

Cell Division ◽

Cell Cycle Arrest ◽

Mitotic Cell ◽

Cycle Arrest ◽

Associated Lesions

AbstractIn Saccharomyces cerevisiae, the conserved Sgs1-Top3-Rmi1 helicase-decatenase regulates homologous recombination by limiting accumulation of recombination intermediates that are precursors of crossovers. In vitro studies have suggested that the dissolution of double-Holliday junction joint molecules by Sgs1-driven convergent junction migration and Top3-Rmi1 mediated strand decatenation could be responsible for this. To ask if dissolution occurs in vivo, we conditionally depleted Sgs1 and/or Rmi1 during return to growth, a procedure where recombination intermediates formed during meiosis are resolved when cells resume the mitotic cell cycle. Sgs1 depletion during return to growth delayed joint molecule resolution, but ultimately most were resolved and cells divided normally. In contrast, Rmi1 depletion resulted in delayed and incomplete joint molecule resolution, and most cells did not divide. rad9Δ mutation restored cell division in Rmi1-depleted cells, indicating that the DNA damage checkpoint caused this cell cycle arrest. Restored cell division in rad9Δ, Rmi1-depleted cells frequently produced anucleate cells, consistent with the suggestion that persistent recombination intermediates prevented chromosome segregation. Our findings indicate that Sgs1-Top3-Rmi1 acts in vivo, as it does in vitro, to promote recombination intermediate resolution by dissolution. They also indicate that, in the absence of Top3-Rmi1 activity, unresolved recombination intermediates persist and activate the DNA damage response, which is usually thought to be activated by much earlier DNA damage-associated lesions.

Download Full-text

Suppression of c-Src activity by C-terminal Src kinase involves the c-Src SH2 and SH3 domains: analysis with Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5290-5300.1993 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5290-5300

Author(s):

S M Murphy ◽

M Bergman ◽

D O Morgan

Keyword(s):

Saccharomyces Cerevisiae ◽

Growth Inhibition ◽

Kinase Activity ◽

Src Kinase ◽

Intact Cells ◽

Sh3 Domains ◽

Mutant Proteins ◽

Dependent Growth

The kinase activity of c-Src is normally repressed in vertebrate cells by extensive phosphorylation of Y-527. C-terminal Src kinase (CSK) is a candidate for the enzyme that catalyzes this phosphorylation. We have used budding yeast to study the regulation of c-Src activity by CSK in intact cells. Expression of c-Src in Saccharomyces cerevisiae, which lacks endogenous c-Src and Y-527 kinases, induces a kinase-dependent growth inhibition. Coexpression of CSK in these cells results in phosphorylation of c-Src on Y-527 and suppression of the c-Src phenotype. CSK does not fully suppress the activity of c-Src mutants lacking portions of the SH2 or SH3 domains, even though these mutant proteins are phosphorylated on Y-527 by CSK both in vivo and in vitro. These results suggest that both the SH2 and SH3 domains of c-Src are required for the suppression of c-Src activity by Y-527 phosphorylation.

Download Full-text

Faithful chromosome transmission requires Spt4p, a putative regulator of chromatin structure in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.16.6.2838 ◽

1996 ◽

Vol 16 (6) ◽

pp. 2838-2847 ◽

Cited By ~ 41

Author(s):

M A Basrai ◽

J Kingsbury ◽

D Koshland ◽

F Spencer ◽

P Hieter

Keyword(s):

Saccharomyces Cerevisiae ◽

Chromosome Instability ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Chromosome Fragment ◽

Dna Mutations ◽

Chromosome Transmission ◽

Biochemical Assays

A chromosome transmission fidelity (ctf) mutant, s138, of Saccharomyces cerevisiae was identified by its centromere (CEN) transcriptional readthrough phenotype, suggesting perturbed kinetochore integrity in vivo. The gene complementing the s138 mutation was found to be identical to the S. cerevisiae SPT4 gene. The s138 mutation is a missense mutation in the second of four conserved cysteine residues positioned similarly to those of zinc finger proteins, and we henceforth refer to the mutation of spt4-138. Both spt4-138 and spt4 delta strains missegregate a chromosome fragment at the permissive temperature, are temperature sensitive for growth at 37 degrees C, and upon a shift to the nonpermissive temperature show an accumulation of large budded cells, each with a nucleus. Previous studies suggest that Spt4p functions in a complex with Spt5p and Spt6p, and we determined that spt6-140 also causes missegregation of a chromosome fragment. Double mutants carrying spt4 delta 2::HIS3 and kinetochore mutation ndc10-42 or ctf13-30 show a synthetic conditional phenotype. Both spt4-138 and spt4 delta strains exhibit synergistic chromosome instability in combination with CEN DNA mutations and show in vitro defects in microtubule binding to minichromosomes. These results indicate that Spt4p plays a role in chromosome segregation. The results of in vivo genetic interactions with mutations in kinetochore proteins and CEN DNA and of in vitro biochemical assays suggest that Spt4p is important for kinetochore function.

Download Full-text

Suppression of c-Src activity by C-terminal Src kinase involves the c-Src SH2 and SH3 domains: analysis with Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5290 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5290-5300 ◽

Cited By ~ 82

Author(s):

S M Murphy ◽

M Bergman ◽

D O Morgan

Keyword(s):

Saccharomyces Cerevisiae ◽

Growth Inhibition ◽

Kinase Activity ◽

Src Kinase ◽

Intact Cells ◽

Sh3 Domains ◽

Mutant Proteins ◽

Dependent Growth

Download Full-text

A Heterodimer of Thioredoxin and IB2 Cooperates with Sec18p (NSF) to Promote Yeast Vacuole Inheritance

The Journal of Cell Biology ◽

10.1083/jcb.136.2.299 ◽

1997 ◽

Vol 136 (2) ◽

pp. 299-306 ◽

Cited By ~ 55

Author(s):

Zuoyu Xu ◽

Andreas Mayer ◽

Eric Muller ◽

William Wickner

Keyword(s):

Saccharomyces Cerevisiae ◽

Early Stage ◽

Redox Chemistry ◽

Soluble Proteins ◽

S Phase ◽

Cell Extracts ◽

Vacuole Inheritance ◽

Cytosolic Inhibitor

Early in S phase, the vacuole (lysosome) of Saccharomyces cerevisiae projects a stream of vesicles and membranous tubules into the bud where they fuse and establish the daughter vacuole. This inheritance reaction can be studied in vitro with isolated vacuoles. Rapid and efficient homotypic fusion between saltwashed vacuoles requires the addition of only two purified soluble proteins, Sec18p (NSF) and LMA1, a novel heterodimer with a thioredoxin subunit. We now report the identity of the second subunit of LMA1 as IB2, a previously identified cytosolic inhibitor of vacuolar proteinase B. Both subunits are needed for efficient vacuole inheritance in vivo and for the LMA1 activity in cell extracts. Each subunit acts via a novel mechanism, as the thioredoxin subunit is not acting through redox chemistry and LMA1 is still needed for the fusion of vacuoles which do not contain proteinase B. Both Sec18p and LMA1 act at an early stage of the in vitro reaction. Though LMA1 does not stimulate Sec18p-mediated Sec17p release, LMA1 cannot fulfill its function before Sec18p. Upon Sec17p/Sec18p action, vacuoles become labile but are rapidly stabilized by LMA1. The action of LMA1 and Sec18p is thus coupled and ordered. These data establish LMA1 as a novel factor in trafficking of yeast vacuoles.

Download Full-text

Incorporation of Ceramides into Saccharomyces cerevisiae Glycosylphosphatidylinositol-Anchored Proteins Can Be Monitored In Vitro

Eukaryotic Cell ◽

10.1128/ec.00257-08 ◽

2008 ◽

Vol 8 (3) ◽

pp. 306-314 ◽

Cited By ~ 8

Author(s):

Régine Bosson ◽

Isabelle Guillas ◽

Christine Vionnet ◽

Carole Roubaty ◽

Andreas Conzelmann

Keyword(s):

Saccharomyces Cerevisiae ◽

Fatty Acid ◽

In Vitro Assay ◽

Gpi Anchor ◽

Intact Cells ◽

Vitro Assay ◽

Gpi Proteins ◽

Mutant Cells

ABSTRACT After glycosylphosphatidylinositols (GPIs) are added to GPI proteins of Saccharomyces cerevisiae, a fatty acid of the diacylglycerol moiety is exchanged for a C26:0 fatty acid through the subsequent actions of Per1 and Gup1. In most GPI anchors this modified diacylglycerol-based anchor is subsequently transformed into a ceramide-containing anchor, a reaction which requires Cwh43. Here we show that the last step of this GPI anchor lipid remodeling can be monitored in microsomes. The assay uses microsomes from cells that have been grown in the presence of myriocin, a compound that blocks the biosynthesis of dihydrosphingosine (DHS) and thus inhibits the biosynthesis of ceramide-based anchors. Such microsomes, when incubated with [3H]DHS, generate radiolabeled, ceramide-containing anchor lipids of the same structure as made by intact cells. Microsomes from cwh43Δ or mcd4Δ mutants, which are unable to make ceramide-based anchors in vivo, do not incorporate [3H]DHS into anchors in vitro. Moreover, gup1Δ microsomes incorporate [3H]DHS into the same abnormal anchor lipids as gup1Δ cells synthesize in vivo. Thus, the in vitro assay of ceramide incorporation into GPI anchors faithfully reproduces the events that occur in mutant cells. Incorporation of [3H]DHS into GPI proteins is observed with microsomes alone, but the reaction is stimulated by cytosol or bovine serum albumin, ATP plus coenzyme A (CoA), or C26:0-CoA, particularly if microsomes are depleted of acyl-CoA. Thus, [3H]DHS cannot be incorporated into proteins in the absence of acyl-CoA.

Download Full-text

Sth1p, a Saccharomyces cerevisiae Snf2p/Swi2p Homolog, Is an Essential ATPase in RSC and Differs From Snf/Swi in Its Interactions With Histones and Chromatin-Associated Proteins

Genetics ◽

10.1093/genetics/150.3.987 ◽

1998 ◽

Vol 150 (3) ◽

pp. 987-1005 ◽

Cited By ~ 2

Author(s):

Jian Du ◽

Irem Nasir ◽

Benjamin K Benton ◽

Michael P Kladde ◽

Brehon C Laurent

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Atp Hydrolysis ◽

Temperature Sensitive ◽

Helicase Domain ◽

Growth Defects ◽

Associated Proteins ◽

2C Dna Content

Abstract The essential Sth1p is the protein most closely related to the conserved Snf2p/Swi2p in Saccharomyces cerevisiae. Sth1p purified from yeast has a DNA-stimulated ATPase activity required for its function in vivo. The finding that Sth1p is a component of a multiprotein complex capable of ATP-dependent remodeling of the structure of chromatin (RSC) in vitro, suggests that it provides RSC with ATP hydrolysis activity. Three sth1 temperature-sensitive mutations map to the highly conserved ATPase/helicase domain and have cell cycle and non-cell cycle phenotypes, suggesting multiple essential roles for Sth1p. The Sth1p bromodomain is required for wild-type function; deletion mutants lacking portions of this region are thermosensitive and arrest with highly elongated buds and 2C DNA content, indicating perturbation of a unique function. The pleiotropic growth defects of sth1-ts mutants imply a requirement for Sth1p in a general cellular process that affects several metabolic pathways. Significantly, an sth1-ts allele is synthetically sick or lethal with previously identified mutations in histones and chromatin assembly genes that suppress snf/swi, suggesting that RSC interacts differently with chromatin than Snf/Swi. These results provide a framework for understanding the ATP-dependent RSC function in modeling chromatin and its connection to the cell cycle.

Download Full-text

Characterization of Cdc47p-minichromosome maintenance complexes in Saccharomyces cerevisiae: identification of Cdc45p as a subunit.

Molecular and Cellular Biology ◽

10.1128/mcb.17.10.5867 ◽

1997 ◽

Vol 17 (10) ◽

pp. 5867-5875 ◽

Cited By ~ 42

Author(s):

S Dalton ◽

B Hopwood

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Dna Replication ◽

Chromosomal Dna ◽

Minichromosome Maintenance ◽

Rate Limiting Step ◽

A Subunit

Cdc47p is a member of the minichromosome maintenance (MCM) family of polypeptides, which have a role in the early stages of chromosomal DNA replication. Here, we show that Cdc47p assembles into stable complexes with two other members of the MCM family, Cdc46p and Mcm3p. The assembly of Cdc47p into complexes with Cdc46p does not appear to be cell cycle regulated, making it unlikely that these interactions per se are a rate-limiting step in the control of S phase. Cdc45p is also shown to interact with Cdc47p in vivo and to be a component of high-molecular-weight MCM complexes in cell lysates. Like MCM polypeptides, Cdc45p is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae; however, Cdc45p remains in the nucleus throughout the cell cycle, whereas MCMs are nuclear only during G1. We characterize two mutations in CDC47 and CDC46 which arrest cells with unduplicated DNA as a result of single base substitutions. The corresponding amino acid substitutions in Cdc46p and Cdc47p severely reduce the ability of these polypeptides to assemble in a complex with each other in vivo and in vitro. This argues that assembly of Cdc47p into complexes with other MCM polypeptides is important for its role in the initiation of chromosomal DNA replication.

Download Full-text