scholarly journals Calcium control of Saccharomyces cerevisiae actin assembly.

1982 ◽  
Vol 2 (10) ◽  
pp. 1279-1286 ◽  
Author(s):  
C Greer ◽  
R Schekman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Speed ◽  
Critical Concentration ◽  
Rabbit Muscle ◽  
Muscle Actin ◽  
Tetraacetic Acid ◽  
Actin Assembly ◽  
Filamentous Actin ◽  
Low Levels

Low levels of Ca2+ dramatically influence the polymerization of Saccharomyces cerevisiae actin in KCl. The apparent critical concentration for polymerization (C infinity) increases eightfold in the presence of 0.1 mM Ca2+. This effect is rapidly reversed by the addition of ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid or of 0.1 mM Mg2+. Furthermore, the addition of Ca2+ to polymerized actin causes a reversible increase in the apparent C infinity. In the presence of Ca2+, at actin concentrations below the apparent C infinity, particles of 15 to 50 nm in diameter are seen instead of filaments. These particles are separated from soluble actin when Ca2+-treated filamentous actin is sedimented at high speed; both the soluble and particulate fractions retain Ca2+-sensitive polymerization. The Ca2+ effect is S. cerevisiae actin-specific: the C infinity for rabbit muscle actin is not affected by the presence of Ca2+ and S. cerevisiae actin. Ca2+ may act directly on S. cerevisiae actin to control the assembly state in vivo.

Calcium control of Saccharomyces cerevisiae actin assembly

1982 ◽  
Vol 2 (10) ◽  
pp. 1279-1286
Author(s):  
C Greer ◽  
R Schekman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Speed ◽  
Critical Concentration ◽  
Rabbit Muscle ◽  
Muscle Actin ◽  
Tetraacetic Acid ◽  
Actin Assembly ◽  
Filamentous Actin ◽  
Low Levels

Low levels of Ca2+ dramatically influence the polymerization of Saccharomyces cerevisiae actin in KCl. The apparent critical concentration for polymerization (C infinity) increases eightfold in the presence of 0.1 mM Ca2+. This effect is rapidly reversed by the addition of ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid or of 0.1 mM Mg2+. Furthermore, the addition of Ca2+ to polymerized actin causes a reversible increase in the apparent C infinity. In the presence of Ca2+, at actin concentrations below the apparent C infinity, particles of 15 to 50 nm in diameter are seen instead of filaments. These particles are separated from soluble actin when Ca2+-treated filamentous actin is sedimented at high speed; both the soluble and particulate fractions retain Ca2+-sensitive polymerization. The Ca2+ effect is S. cerevisiae actin-specific: the C infinity for rabbit muscle actin is not affected by the presence of Ca2+ and S. cerevisiae actin. Ca2+ may act directly on S. cerevisiae actin to control the assembly state in vivo.

Actin from Saccharomyces cerevisiae

1982 ◽  
Vol 2 (10) ◽  
pp. 1270-1278
Author(s):  
C Greer ◽  
R Schekman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Sodium Dodecyl ◽  
Rabbit Muscle ◽  
Muscle Actin ◽  
Filamentous Actin ◽  
Heavy Meromyosin ◽  
Proteolytic Fragment ◽  
Maximal Activation ◽  
Final Fraction ◽  
Muscle Myosin

Inhibition of DNase I activity has been used as an assay to purify actin from Saccharomyces cerevisiae (yeast actin). The final fraction, obtained after a 300-fold purification, is approximately 97% pure as judged by sodium dodecyl sulfate-gel electrophoresis. Like rabbit skeletal muscle actin, yeast actin has a molecular weight of about 43,000, forms 7-nm-diameter filaments when polymerization is induced by KCl or Mg2+, and can be decorated with a proteolytic fragment of muscle myosin (heavy meromyosin). Although heavy meromyosin ATPase activity is stimulated by rabbit muscle and yeast actins to approximately the same Vmax (2 mmol of Pi per min per mumol of heavy meromyosin), half-maximal activation (Kapp) is obtained with 14 micro M muscle actin, but requires approximately 135 micro M yeast actin. This difference suggests a low affinity of yeast actin for muscle myosin. Yeast and muscle filamentous actin respond similarly to cytochalasin and phalloidin, although the drugs have no effect on S. cerevisiae cell growth.

scholarly journals Actin from Saccharomyces cerevisiae.

1982 ◽  
Vol 2 (10) ◽  
pp. 1270-1278 ◽  
Author(s):  
C Greer ◽  
R Schekman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Sodium Dodecyl ◽  
Rabbit Muscle ◽  
Muscle Actin ◽  
Filamentous Actin ◽  
Heavy Meromyosin ◽  
Proteolytic Fragment ◽  
Maximal Activation ◽  
Final Fraction ◽  
Muscle Myosin

Inhibition of DNase I activity has been used as an assay to purify actin from Saccharomyces cerevisiae (yeast actin). The final fraction, obtained after a 300-fold purification, is approximately 97% pure as judged by sodium dodecyl sulfate-gel electrophoresis. Like rabbit skeletal muscle actin, yeast actin has a molecular weight of about 43,000, forms 7-nm-diameter filaments when polymerization is induced by KCl or Mg2+, and can be decorated with a proteolytic fragment of muscle myosin (heavy meromyosin). Although heavy meromyosin ATPase activity is stimulated by rabbit muscle and yeast actins to approximately the same Vmax (2 mmol of Pi per min per mumol of heavy meromyosin), half-maximal activation (Kapp) is obtained with 14 micro M muscle actin, but requires approximately 135 micro M yeast actin. This difference suggests a low affinity of yeast actin for muscle myosin. Yeast and muscle filamentous actin respond similarly to cytochalasin and phalloidin, although the drugs have no effect on S. cerevisiae cell growth.

A nuclear gene of Saccharomyces cerevisiae needed for stable maintenance of plasmids

1986 ◽  
Vol 6 (11) ◽  
pp. 4053-4059
Author(s):  
Y Kikuchi ◽  
A Toh-e
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Gene ◽  
Complementation Group ◽  
Chromosomal Replication ◽  
Autonomously Replicating Sequence ◽  
Initiation Of Replication ◽  
Low Levels ◽  
Stable Maintenance

We have isolated host mutants of Saccharomyces cerevisiae in which the 2 microns plasmid is poorly maintained. All the mutants tested constituted one complementation group, which was designated map1 (maintenance of plasmid). Minichromosomes carrying a chromosomal replication origin and a centromere were affected in the mutants. Two types of hybrid plasmids generated in vivo and in vitro appeared to compensate for the mutations and had DNA regions containing multiple ARS (autonomously replicating sequence) or a set of 2 microns inverted repeat sequences. These results suggested that poor maintenance of plasmids was due to low levels of replication, probably at the initiation of replication.

scholarly journals A nuclear gene of Saccharomyces cerevisiae needed for stable maintenance of plasmids.

1986 ◽  
Vol 6 (11) ◽  
pp. 4053-4059 ◽  
Author(s):  
Y Kikuchi ◽  
A Toh-e
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Gene ◽  
Complementation Group ◽  
Chromosomal Replication ◽  
Autonomously Replicating Sequence ◽  
Initiation Of Replication ◽  
Low Levels ◽  
Stable Maintenance

We have isolated host mutants of Saccharomyces cerevisiae in which the 2 microns plasmid is poorly maintained. All the mutants tested constituted one complementation group, which was designated map1 (maintenance of plasmid). Minichromosomes carrying a chromosomal replication origin and a centromere were affected in the mutants. Two types of hybrid plasmids generated in vivo and in vitro appeared to compensate for the mutations and had DNA regions containing multiple ARS (autonomously replicating sequence) or a set of 2 microns inverted repeat sequences. These results suggested that poor maintenance of plasmids was due to low levels of replication, probably at the initiation of replication.

scholarly journals Actin activatesPseudomonas aeruginosaExoY nucleotidyl cyclase toxin and ExoY-like effector domains from MARTX toxins

2015 ◽  
Author(s):  
Dorothee Raoux-Barbot ◽  
Cosmin Saveanu ◽  
Abdelkader Namane ◽  
Vasily Ogryzko ◽  
Lina Worpenberg ◽  
...  
Keyword(s):  
Actin Binding ◽  
Target Cells ◽  
Actin Assembly ◽  
Filamentous Actin ◽  
Severe Damage ◽  
Chronic Infections ◽  
Effector Domains ◽  
Host Target

Pseudomonas aeruginosa is a major cause of chronic infections in cystic fibrosis patients. The nucleotidyl cyclase toxin ExoY is a virulence factors injected by the pathogen and associated with severe damage to lung tissue. ExoY-like cyclases are also found in other Gram-negative pathogens and shown to contribute to virulence, although they remained poorly characterized. Here we demonstrate that filamentous actin (F-actin) is the hitherto unknown co-factor that activates P. aeruginosa ExoY within host target cells. Highly purified actin, when polymerized into filaments, potently stimulates (>10,000 fold) ExoY activity. ExoY co-localizes in vivo with actin filaments in transfected cells and, in vitro, it interferes with the regulation of actin assembly/disassembly-dynamics mediated by important F-actin-binding proteins. We further show that actin also activates an ExoY-like adenylate cyclase from a Vibrio species. Our results thus highlight a new sub-class within the class II adenylyl cyclase family, defined as actin-activated nucleotidyl cyclase (AA-NC) toxins.

scholarly journals Regulation of Cell Polarity by Interactions of Msb3 and Msb4 with Cdc42 and Polarisome Components

2005 ◽  
Vol 25 (19) ◽  
pp. 8567-8580 ◽  
Author(s):  
Serguei E. Tcheperegine ◽  
Xiang-Dong Gao ◽  
Erfei Bi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Polarity ◽  
Rab Gtpase ◽  
Polarized Growth ◽  
Actin Assembly ◽  
Gtpase Activating Proteins ◽  
Functional Equivalent ◽  
Gdp Dissociation Inhibitor

ABSTRACT In Saccharomyces cerevisiae, polarized growth depends on interactions between the actin cytoskeleton and the secretory machinery. Here we show that the Rab GTPase-activating proteins (GAPs) Msb3 and Msb4 interact directly with Spa2, a scaffold protein of the “polarisome” that also interacts with the formin Bni1. Spa2 is required for the polarized localization of Msb3 and Msb4 at the bud tip. We also show that Msb3 and Msb4 bind specifically to Cdc42-GDP and Rho1-GDP in vitro and that Msb3 and Rho GDP dissociation inhibitor act independently but oppositely on Cdc42. Finally, we show that Msb3 and Msb4 are involved in Bni1-nucleated actin assembly in vivo. These results suggest that Msb3 and Msb4 regulate polarized growth by multiple mechanisms, directly regulating exocytosis through their GAP activity toward Sec4 and potentially coordinating the functions of Cdc42, Rho1, and Bni1 in the polarisome through their binding to these GTPases. A functional equivalent of the polarisome probably exists in other fungi and mammals.

scholarly journals Actin from Thyone sperm assembles on only one end of an actin filament: a behavior regulated by profilin.

1983 ◽  
Vol 97 (1) ◽  
pp. 112-124 ◽  
Author(s):  
L G Tilney ◽  
E M Bonder ◽  
L M Coluccio ◽  
M S Mooseker
Keyword(s):  
Isoelectric Point ◽  
Actin Filaments ◽  
Critical Concentration ◽  
Molar Ratio ◽  
Actin Assembly ◽  
Actin Monomer ◽  
Filament Bundles ◽  
Sperm Extract ◽  
Triton X

Thyone sperm were demembranated with Triton X-100 and, after washing, extracted with 30 mM Tris at pH 8.0 and 1 mM MgCl2. After the insoluble contaminants were removed by centrifugation, the sperm extract was warmed to 22 degrees C. Actin filaments rapidly assembled and aggregated into bundles when KCl was added to the extract. When we added preformed actin filaments, i.e., the acrosomal filament bundles of Limulus sperm, to the extract, the actin monomers rapidly assembled on these filaments. What was unexpected was that assembly took place on only one end of the bundle--the end corresponding to the preferred end for monomer addition. We showed that the absence of growth on the nonpreferred end was not due to the presence of a capper because exogenously added actin readily assembled on both ends. We also analyzed the sperm extract by SDS gel electrophoresis. Two major proteins were present in a 1:1 molar ratio: actin and a 12,500-dalton protein whose apparent isoelectric point was 8.4. The 12,500-dalton protein was purified by DEAE chromatography. We concluded that it is profilin because of its size, isoelectric point, molar ratio to actin, inability to bind to DEAE, and its effect on actin assembly. When profilin was added to actin in the presence of Limulus bundles, addition of monomers on the nonpreferred end of the bundle was inhibited, even though actin by itself assembled on both ends. Using the Limulus bundles as nuclei, we determined the critical concentration for assembly off each end of the filament and estimated the Kd for the profilin-actin complex (approximately 10 microM). We present a model to explain how profilin may regulate the extension of the Thyone acrosomal process in vivo: The profilin-actin complex can add to only the preferred end of the filament bundle. Once the actin monomer is bound to the filament, the profilin is released, and is available to bind to additional actin monomers. This mechanism accounts for the rapid rate of filament elongation in the acrosomal process in vivo.

scholarly journals Phalloidin enhances actin assembly by preventing monomer dissociation.

1984 ◽  
Vol 99 (2) ◽  
pp. 529-535 ◽  
Author(s):  
L M Coluccio ◽  
L G Tilney
Keyword(s):  
Rate Constant ◽  
Actin Filaments ◽  
Critical Concentration ◽  
Dissociation Rate ◽  
Dissociation Rate Constant ◽  
Muscle Actin ◽  
Actin Assembly ◽  
Association Rate Constant ◽  
Association Rate ◽  
Dissociation Rate Constants

Incubation of the isolated acrosomal bundles of Limulus sperm with skeletal muscle actin results in assembly of actin onto both ends of the bundles. These cross-linked bundles of actin filaments taper, thus allowing one to distinguish directly the preferred end for actin assembly from the nonpreferred end; the preferred end is thinner. Incubation with actin in the presence of equimolar phalloidin in 100 mM KCl, 1 mM MgCl2 and 0.5 mM ATP at pH 7.5 resulted in a slightly smaller association rate constant at the preferred end than in the absence of the drug (3.36 +/- 0.14 X 10(6) M-1 s-1 vs. 2.63 +/- 0.22 X 10(6) M-1 s-1, control vs. experimental). In the presence of phalloidin, the dissociation rate constant at the preferred end was reduced from 0.317 +/- 0.097 s-1 to essentially zero. Consequently, the critical concentration at the preferred end dropped from 0.10 microM to zero in the presence of the drug. There was no detectable change in the rate constant of association at the nonpreferred end in the presence of phalloidin (0.256 +/- 0.015 X 10(6) M-1 s-1 vs. 0.256 +/- 0.043 X 10(6) M-1 s-1, control vs. experimental); however, the dissociation rate constant was reduced from 0.269 +/- 0.043 s-1 to essentially zero. Thus, the critical concentration at the nonpreferred end changed from 1.02 microM to zero in the presence of phalloidin. Dilution-induced depolymerization at both the preferred and nonpreferred ends was prevented in the presence of phalloidin. Thus, phalloidin enhances actin assembly by lowering the critical concentration at both ends of actin filaments, a consequence of reducing the dissociation rate constants at each end.

scholarly journals The Iron-Sulfur Cluster Proteins Isa1 and Isa2 Are Required for the Function but Not for the De Novo Synthesis of the Fe/S Clusters of Biotin Synthase in Saccharomyces cerevisiae

2007 ◽  
Vol 6 (3) ◽  
pp. 495-504 ◽  
Author(s):  
Ulrich Mühlenhoff ◽  
Mathias J. Gerl ◽  
Birgit Flauger ◽  
Heike M. Pirner ◽  
Sandra Balser ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Mitochondrial Protein ◽  
Cluster Assembly ◽  
De Novo Synthesis ◽  
Biotin Synthase ◽  
Iron Sulfur Cluster ◽  
Iron Sulfur ◽  
Low Levels

ABSTRACT The yeast Saccharomyces cerevisiae is able to use some biotin precursors for biotin biosynthesis. Insertion of a sulfur atom into desthiobiotin, the final step in the biosynthetic pathway, is catalyzed by biotin synthase (Bio2). This mitochondrial protein contains two iron-sulfur (Fe/S) clusters that catalyze the reaction and are thought to act as a sulfur donor. To identify new components of biotin metabolism, we performed a genetic screen and found that Isa2, a mitochondrial protein involved in the formation of Fe/S proteins, is necessary for the conversion of desthiobiotin to biotin. Depletion of Isa2 or the related Isa1, however, did not prevent the de novo synthesis of any of the two Fe/S centers of Bio2. In contrast, Fe/S cluster assembly on Bio2 strongly depended on the Isu1 and Isu2 proteins. Both isa mutants contained low levels of Bio2. This phenotype was also found in other mutants impaired in mitochondrial Fe/S protein assembly and in wild-type cells grown under iron limitation. Low Bio2 levels, however, did not cause the inability of isa mutants to utilize desthiobiotin, since this defect was not cured by overexpression of BIO2. Thus, the Isa proteins are crucial for the in vivo function of biotin synthase but not for the de novo synthesis of its Fe/S clusters. Our data demonstrate that the Isa proteins are essential for the catalytic activity of Bio2 in vivo.

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