Budding cells of the yeast Saccharomyces cerevisiae possess a ring 10-nm-diameter filaments, unknown biochemical nature, that lies just inside plasma membrane in neck connecting mother cell to its bud. Electron microscopic observations suggest these filaments assemble at budding site coincident with bud emergence and disassemble shortly before cytokinesis (Byers, B. L. Goetsch. 1976. J. Cell Biol. 69:717-721). Mutants defective any four genes (CDC3, CDC10, CDC11, or CDC12) lack display...

Budding cells of the yeast Saccharomyces cerevisiae possess a ring 10-nm-diameter filaments, unknown biochemical nature, that lies just inside plasma membrane in neck connecting mother cell to its bud (B. Byers and L. Goetsch, J. Cell Biol. 69:717-721, 1976). Mutants defective any four genes (CDC3, CDC10, CDC11, CDC12) lack these filaments display pleiotropic phenotype involves abnormal growth cell-wall deposition an inability complete cytokinesis. We fused cloned CDC12 gene Escherichia coli...

We have isolated profilin from yeast (Saccharomyces cerevisiae) and microsequenced a portion of the protein to confirm its identity; region agrees with predicted amino acid sequence gene recently S. cerevisiae (Magdolen, V., U. Oechsner, G. Müller, W. Bandlow. 1988. Mol. Cell. Biol. 8:5108-5115). Yeast resembles profilins other organisms in molecular mass ability bind polyproline, retard rate actin polymerization, inhibit hydrolysis ATP by monomeric actin. Using strains that carry...

The septins are GTP-binding, filament-forming proteins that involved in cytokinesis and other processes. In the yeast Saccharomyces cerevisiae, recruited to presumptive bud site at cell cortex, where they form a ring through which emerges. We report here wild-type cells, typically become detectable vicinity of several minutes before formation, but itself is first distinct structure forms. Septin recruitment depends on activated Cdc42p not normal pathway for bud-site selection. Recruitment...

We have isolated a fourth myosin gene (MYO4) in yeast (Saccharomyces cerevisiae). MYO4 encodes approximately 170 kDa (1471 amino acid) class V myosin, using the classification devised by Cheney et al. (1993a; Cell Motil. Cytoskel. 24, 215-223); motor domain is followed neck region containing six putative calmodulin-binding sites and tail with short potential 'coiled-coil' domain. A comparison other myosins GenBank reveals that Myo4 protein most closely related to Myo2 protein, another...

Abstract Actin oxidation is known to result in changes cytoskeleton organization and dynamics. clinically relevant since it occurs the erythrocytes of sickle cell patients may be direct cause lack morphological plasticity observed irreversibly sickled red blood cells (ISCs). During episodes crisis, ISCs accumulate C284‐C373 intramolecularly disulfide bonded actin, which reduces actin filament cysteines 284 373 (285 374 yeast) are conserved, suggesting that they play an important functional...

We have previously demonstrated that in Saccharomyces cerevisiae replication, checkpoint inactivation via a mec1 mutation leads to chromosome breakage at replication forks initiated from virtually all origins after transient exposure hydroxyurea (HU), an inhibitor of ribonucleotide reductase. Here we sought determine whether containing single-stranded DNA gaps equal probability producing double-strand breaks (DSBs) when cells attempt recover HU exposure. devised new methodology, Break-seq,...

We have mutated two regions within the yeast profilin gene in an effort to functionally dissect roles of actin and phosphatidylinositol 4,5-bisphosphate (PIP2) binding function. A series truncations was carried out at C terminus profilin, a region that has been implicated binding. Removal last three amino acids nearly eliminated ability bind polyproline vitro but had no dramatic vivo effects. Thus, extreme is binding, physiological relevance this interaction called into question. More...

Budding cells of the yeast Saccharomyces cerevisiae possess a ring 10-nm-diameter filaments, unknown biochemical nature, that lies just inside plasma membrane in neck connecting mother cell to its bud (B. Byers and L. Goetsch, J. Cell Biol. 69:717-721, 1976). Mutants defective any four genes (CDC3, CDC10, CDC11, CDC12) lack these filaments display pleiotropic phenotype involves abnormal growth cell-wall deposition an inability complete cytokinesis. We fused cloned CDC12 gene Escherichia coli...

Multigenic influences are major contributors to human genetic disorders. Since humans highly polymorphic, there a high number of possible detrimental, multiallelic gene pairs. The actin cytoskeleton yeast was used determine the potential for deleterious bigenic interactions; ∼4800 complex hemizygote strains were constructed between an actin-null allele and nonessential deletion collection. We found 208 genes that have haploinsufficient (CHI) interactions with actin. This set is enriched...

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by mutations in the FMR1 gene and deficiency of functional FMRP protein. known as translation repressor whose nuclear function not understood. We investigated global impact on genome stability due to loss. Using Break-seq, we map spontaneous replication stress-induced DNA double-strand breaks (DSBs) an FXS patient-derived cell line. report that genomes cells are inherently unstable accumulate twice many DSBs those from...

Cell processes require precise regulation of actin polymerization that is mediated by plus-end regulatory proteins. Detailed mechanisms explain dynamics involve regulators with opposing roles, including factors enhance assembly, e.g., the formin mDia1, and others stop growth (capping protein, CP). We explore IQGAP1’s roles in regulating filament plus-ends consequences perturbing its activity cells. confirm IQGAP1 pauses elongation interacts plus ends through two residues (C756 C781)....

The structure of profilin from the budding yeast Saccharomyces cerevisiae has been determined by X-ray crystallography at 2.3 Å resolution. overall fold is similar to observed for other structures. interactions and human platelet profilins with rabbit skeletal muscle actin were characterized titration microcalorimetry, fluorescence titrations, nucleotide exchange kinetics. affinity (2.9 μM) approximately 30-fold weaker than (0.1 μM), relative contributions entropic enthalpic terms free...

F1-ATPase is the catalytic complex of rotary nanomotor ATP synthases. Bacterial synthases can be autoinhibited by C-terminal domain subunit ε, which partially inserts into enzyme's central rotor cavity to block functional rotation. Using a kinetic, optical assay F1·ε binding and dissociation, we show that formation extended, inhibitory conformation ε (εX) initiates after hydrolysis at dwell step. Prehydrolysis conditions prevent εX state, post-hydrolysis stabilize it. We also inhibition ADP...

Eight separate mutations in the actin-binding protein profilin-1 have been identified as a rare cause of amyotrophic lateral sclerosis (ALS). Profilin is essential for many neuronal cell processes through its regulation lipids, nuclear signals, and cytoskeletal dynamics, including actin filament assembly. Direct interactions between profilin monomers inhibit polymerization. In contrast, can also stimulate polymerization by simultaneously binding proline-rich tracts found other proteins....

Old Yellow Enzyme (OYE) has long served as a paradigm for the study of flavin-containing NADPH oxido-reductases and yet its physiological role remained mystery. A two-hybrid interaction between Oye2p actin led us to investigate possible function in cytoskeleton. We found that oye deletion strains have an overly elaborate cytoskeleton cannot be attributed changes concentration but likely reflect stabilization filaments, resulting excessive assembly. Cells expressing mutant act1-123p, which...

Replacement of the wild-type yeast profilin gene (PFY1) with a mutated form (pfy1-111) that has codon 72 changed to encode glutamate rather than arginine results in defects similar to, but less severe than, those result from complete deletion gene. We have used colony color-sectoring assay identify mutations cause pfy1-111, not wild-type, cells be inviable. These synthetic lethal (psl) various degrees abnormal growth, morphology, and temperature sensitivity PFY1 cells. examined psl1 strains...

In the yeast, <i>Saccharomyces cerevisiae</i>, adenylyl cyclase consists of a 200-kDa catalytic subunit (<i>CYR1</i>) and 70-kDa (<i>CAP/SRV2</i>). CAP/Srv2p assists small G protein Ras to activate cyclase. CAP also regulates cytoskeleton through an actin sequestering activity is directed cortical patches by proline-rich SH3-binding site (P₂). this report we analyze role in Ras/cAMP signaling. Two alleles CAP, L16P(Srv2) R19T (SupC), first isolated genetic screens for mutants that...

Actin interacting protein 1 (Aip1p) and cofilin cooperate to disassemble actin filaments in vitro are thought promote rapid turnover of networks vivo. The precise method by which Aip1p participates these activities has not been defined, although severing barbed-end capping have proposed. To better describe the mechanisms biological consequences activities, we undertook an extensive mutagenesis AIP1 aimed at disrupting mapping interactions. Site-directed suggested that two binding sites,...

Saccharomyces cerevisiae has been a powerful model for uncovering the landscape of binary gene interactions through whole-genome screening. Complex heterozygous are potentially important to human genetic disease as loss-of-function alleles common in genomes. We have using complex haploinsufficiency (CHI) screening with actin identify genes related function and determine prevalence CHI eukaryotic Previous between null non-essential uncovered ∼240 deleterious interactions. In this report, we...

We have mutated two regions within the yeast profilin gene in an effort to functionally dissect roles of actin and phosphatidylinositol 4,5-bisphosphate (PIP2) binding function. A series truncations was carried out at C terminus profilin, a region that has been implicated binding. Removal last three amino acids nearly eliminated ability bind polyproline vitro but had no dramatic vivo effects. Thus, extreme is binding, physiological relevance this interaction called into question. More...