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E Bi

Publications and source records attributed to E Bi.

At least 19 recordsLinked to original sources
PubMed2001

Cdc42 interacts with the exocyst and regulates polarized secretion.

Polarized delivery and incorporation of proteins and lipids to specific domains of the plasma membrane is fundamental to a wide range of biological processes such as neuronal synaptogenesis and epithelial cell polarization. The exocyst complex is specifically localized to sites of active exocytosis and plays essential roles in secretory vesicle targeting and docking at the plasma membrane. Sec3p, a component of the exocyst, is thought to be a spatial landmark for polarized exocytosis. In a search for proteins that regulate the localization of the exocyst in the budding yeast Saccharomyces cerevisiae, we found that certain cdc42 mutants affect the polarized localization of the exocyst proteins. In addition, we found that these mutant cells have a randomized protein secretion pattern on the cell surface. Biochemical experiments indicated that Sec3p directly interacts with Cdc42 in its GTP-bound form. Genetic studies demonstrated synthetically lethal interactions between cdc42 and several exocyst mutants. These results have revealed a role for Cdc42 in exocytosis. We propose that Cdc42 coordinates the vesicle docking machinery and the actin cytoskeleton for polarized secretion.

Actins↗
PubMed2001

A protein interaction map for cell polarity development.

Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes. To enumerate more fully the protein-protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express approximately 90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions. 191 protein-protein interactions were detected, of which 128 had not been described previously. 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development. Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed.

Actins↗
PubMed2001

Cytokinesis in budding yeast: the relationship between actomyosin ring function and septum formation.

Cytokinesis in budding yeast is accomplished by the concerted action of actomyosin ring function and septum formation. The actomyosin ring is not essential for cell viability, but it is required for efficient cell division. Deletion of the actomyosin ring results in abnormal septum formation, and a delay in cytokinesis and cell separation. In contrast, septum formation is essential for cell viability. Block of septum formation prevents the contraction, but not the formation of the actomyosin ring. Here we review and provide additional evidence that defines the functional and molecular relationship between actomyosin ring function and septum formation.

Actins↗
PubMed2000

Roles of Hof1p, Bni1p, Bnr1p, and myo1p in cytokinesis in Saccharomyces cerevisiae.

Cytokinesis in Saccharomyces cerevisiae occurs by the concerted action of the actomyosin system and septum formation. Here we report on the roles of HOF1, BNI1, and BNR1 in cytokinesis, focusing on Hof1p. Deletion of HOF1 causes a temperature-sensitive defect in septum formation. A Hof1p ring forms on the mother side of the bud neck in G2/M, followed by the formation of a daughter-side ring. Around telophase, Hof1p is phosphorylated and the double rings merge into a single ring that contracts slightly and may colocalize with the actomyosin structure. Upon septum formation, Hof1p splits into two rings, disappearing upon cell separation. Hof1p localization is dependent on septins but not Myo1p. Synthetic lethality suggests that Bni1p and Myo1p belong to one functional pathway, whereas Hof1p and Bnr1p belong to another. These results suggest that Hof1p may function as an adapter linking the primary septum synthesis machinery to the actomyosin system. The formation of the actomyosin ring is not affected by bni1Delta, hof1Delta, or bnr1Delta. However, Myo1p contraction is affected by bni1Delta but not by hof1Delta or bnr1Delta. In bni1Delta cells that lack the actomyosin contraction, septum formation is often slow and asymmetric, suggesting that actomyosin contraction may provide directionality for efficient septum formation.

Actins↗
PubMed2000

Identification of novel, evolutionarily conserved Cdc42p-interacting proteins and of redundant pathways linking Cdc24p and Cdc42p to actin polarization in yeast.

In the yeast Saccharomyces cerevisiae, Cdc24p functions at least in part as a guanine-nucleotide-exchange factor for the Rho-family GTPase Cdc42p. A genetic screen designed to identify possible additional targets of Cdc24p instead identified two previously known genes, MSB1 and CLA4, and one novel gene, designated MSB3, all of which appear to function in the Cdc24p-Cdc42p pathway. Nonetheless, genetic evidence suggests that Cdc24p may have a function that is distinct from its Cdc42p guanine-nucleotide-exchange factor activity; in particular, overexpression of CDC42 in combination with MSB1 or a truncated CLA4 in cells depleted for Cdc24p allowed polarization of the actin cytoskeleton and polarized cell growth, but not successful cell proliferation. MSB3 has a close homologue (designated MSB4) and two more distant homologues (MDR1 and YPL249C) in S. cerevisiae and also has homologues in Schizosaccharomyces pombe, Drosophila (pollux), and humans (the oncogene tre17). Deletion of either MSB3 or MSB4 alone did not produce any obvious phenotype, and the msb3 msb4 double mutant was viable. However, the double mutant grew slowly and had a partial disorganization of the actin cytoskeleton, but not of the septins, in a fraction of cells that were larger and rounder than normal. Like Cdc42p, both Msb3p and Msb4p localized to the presumptive bud site, the bud tip, and the mother-bud neck, and this localization was Cdc42p dependent. Taken together, the data suggest that Msb3p and Msb4p may function redundantly downstream of Cdc42p, specifically in a pathway leading to actin organization. From previous work, the BNI1, GIC1, and GIC2 gene products also appear to be involved in linking Cdc42p to the actin cytoskeleton. Synthetic lethality and multicopy suppression analyses among these genes, MSB, and MSB4, suggest that the linkage is accomplished by two parallel pathways, one involving Msb3p, Msb4p, and Bni1p, and the other involving Gic1p and Gic2p. The former pathway appears to be more important in diploids and at low temperatures, whereas the latter pathway appears to be more important in haploids and at high temperatures.

Actins↗
PubMed1999

Actin polymerization: Where the WASP stings.

How do extracellular signals induce actin polymerization, as required for many cellular responses? Key signal transducers, such as the small GTPases Cdc42 and Rac, have now been shown to link via proteins of the WASP family to the Arp2/3 complex, which nucleates actin polymerization.

Actin-Related Protein 2↗
PubMed1999

Cytokinesis: an emerging unified theory for eukaryotes?

In animal and fungal cells, cytokinesis involves an actomyosin ring that forms and contracts at the division plane. Important new details have emerged concerning the composition, assembly, and dynamics of these contractile rings. In addition, recent advances suggest that targeted membrane addition is a central feature of cytokinesis in animal cells - as it is in fungi and plants - and the coordination of actomyosin ring function with targeted exocytosis at the cleavage plane is being explored. Important new information has also emerged about the spatial and temporal regulation of cytokinesis, especially in relation to the function of the spindle midzone in animal cells and the control of cytokinesis by GTPase systems.

Animals↗
PubMed1998

Involvement of an actomyosin contractile ring in Saccharomyces cerevisiae cytokinesis.

In Saccharomyces cerevisiae, the mother cell and bud are connected by a narrow neck. The mechanism by which this neck is closed during cytokinesis has been unclear. Here we report on the role of a contractile actomyosin ring in this process. Myo1p (the only type II myosin in S. cerevisiae) forms a ring at the presumptive bud site shortly before bud emergence. Myo1p ring formation depends on the septins but not on F-actin, and preexisting Myo1p rings are stable when F-actin is depolymerized. The Myo1p ring remains in the mother-bud neck until the end of anaphase, when a ring of F-actin forms in association with it. The actomyosin ring then contracts to a point and disappears. In the absence of F-actin, the Myo1p ring does not contract. After ring contraction, cortical actin patches congregate at the mother-bud neck, and septum formation and cell separation rapidly ensue. Strains deleted for MYO1 are viable; they fail to form the actin ring but show apparently normal congregation of actin patches at the neck. Some myo1Delta strains divide nearly as efficiently as wild type; other myo1Delta strains divide less efficiently, but it is unclear whether the primary defect is in cytokinesis, septum formation, or cell separation. Even cells lacking F-actin can divide, although in this case division is considerably delayed. Thus, the contractile actomyosin ring is not essential for cytokinesis in S. cerevisiae. In its absence, cytokinesis can still be completed by a process (possibly localized cell-wall synthesis leading to septum formation) that appears to require septin function and to be facilitated by F-actin.

Actins↗
PubMed1998

Elevated expression of chitinase 1 and chitin synthesis in myosin II-deficient Saccharomyces cerevisiae.

To determine if the attached cells formed in Myosin II-deficient Saccharomyces cerevisiae result from deficient chitinase 1 (CTS1) expression, the activity of chitinase 1 was assayed. Secretion of this enzyme was not prevented by a MYO1 gene deficiency, and soluble and cell wall-associated Cts1p activity were increased approximately 5-fold and 20-fold, respectively, in these cells. The increase in soluble activity was correlated with an increase in enzyme levels. Likewise, intracellular chitinase activity was increased approximately 22-fold, and the chitin content of cell walls was elevated 2-fold. These data suggest that the origin of myo1-associated phenotypes is not due to deficient chitinase expression and may instead be due to a deregulation of cell wall metabolism in these cells.

Cell Wall↗
PubMed1997

Two active states of the Ras-related Bud1/Rsr1 protein bind to different effectors to determine yeast cell polarity.

Cells of budding yeast organize their cytoskeleton in a highly polarized manner during vegetative growth. Selection of a site for polarization requires a group of proteins including a Ras-like GTPase, Bud1, and its regulators. Another group of proteins, which includes a Rho-like GTPase (Cdc42), its guanine nucleotide exchange factor (Cdc24), and Bem1, is necessary for organization of the actin cytoskeleton and for cell polarization. We have proposed previously that the Bud1 protein, through its GTPase cycle, determines the localization of one or more of the cell polarity proteins to the bud site. Herein we demonstrate that Bud1 directly interacts with Cdc24 and Bem1: Bud1 in its GTP-bound form associates preferentially with Cdc24, whereas the GDP-bound form of Bud1 associates with Bem1. We also present subcellular fractionation data for Bud1 that is consistent with the idea that Bud1 can travel between the site for budding on the plasma membrane and the cytosol. We propose that Bud1 can exist in two active states for association with different partners and that the switch from Bud1-GTP to Bud1-GDP provides a regulatory device for ordered assembly of a macromolecular complex at the bud site.

Adaptor Proteins, Signal Transducing↗
PubMed1996

FtsZ ring formation in fts mutants.

The formation of FtsZ rings (Z rings) in various fts mutants was examined by immunoelectron microscopy and immunofluorescence. In two temperature-sensitive ftsZ mutants which form filaments with smooth morphology, the Z ring was unable to form. In ftsA, ftsI, and ftsQ mutants, which form filaments with an indented morphology, Z rings formed but their contraction was blocked. These results indicate that fully functional ftsA, ftsQ, and ftsI genes are not required for Z-ring formation and are unlikely to have a role in localization of the Z ring. The results also suggest that one function of the Z ring is to localize the activity of other fts gene products.

Bacterial Proteins↗
PubMed1996

ZDS1 and ZDS2, genes whose products may regulate Cdc42p in Saccharomyces cerevisiae.

A genetic screen for GTPase-activating proteins (GAPs) or other negative regulators of the Rac/Rho family GTPase Cdc42p in Saccharomyces cerevisiae identified ZDS1, a gene encoding a protein of 915 amino acids. Sequence from the yeast genome project identified a homolog, ZDS2, whose predicted product of 942 amino acids is 38% identical in sequence to Zds1p. Zds1p and Zds2p have no detectable homology to known Rho-GAPs or to other known proteins. However, by several assays, it appears that overexpression of either Zds1p or Zds2p decreases the level of Cdc42p activity. Deletion analysis also suggests that Zds1p and Zds2p are at least partially overlapping in function. Deletion of ZDS2 produced no obvious phenotype, and deletion of ZDS1 produced no obvious phenotype other than a mild effect on cell shape. However, the zds1 zds2 double mutant grew slowly with an apparent mitotic delay and produced elongated cells and buds with other evidence of abnormal morphogenesis. A glutathione S-transferase-Zds1p fusion protein that fully complemented the double mutant localized to presumptive bud sites and the tips of small buds. The similarity of this localization to that of Cdc42p suggests that Zds1p may interact directly with Cdc42p. As ZDS1 and ZDS2 have recently been identified also by numerous other groups studying a wide range of biological phenomena, the roles of Cdc42p in intracellular signaling may be more diverse than has previously been appreciated.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Mutation of RGA1, which encodes a putative GTPase-activating protein for the polarity-establishment protein Cdc42p, activates the pheromone-response pathway in the yeast Saccharomyces cerevisiae.

We have selected yeast mutants that exhibit a constitutively active pheromone-response pathway in the absence of the beta subunit of the trimeric G protein. Genetic analysis of one such mutant revealed that it contained recessive mutations in two distinct genes, both of which contributed to the constitutive phenotype. One mutation identifies the RGA1 locus (Rho GTPase activating protein), which encodes a protein with homology to GAP domains and to LIM domains. Deletion of RGA1 is sufficient to activate the pathway in strains lacking the G beta subunit. Moreover, in wild-type strains, deletion of RGA1 increases signaling in the pheromone pathway, whereas over-expression of RGA1 dampens signaling, demonstrating that Rga1p functions as a negative regulator of the pheromone response pathway. The second mutation present in the original mutant proved to be an allele of a known gene, PBS2, which encodes a putative protein kinase that functions in the high osmolarity stress pathway. The pbs2 mutation enhanced the rga1 mutant phenotype, but by itself did not activate the pheromone pathway. Genetic and two-hybrid analyses indicate that an important target of Rga1p is Cdc42p, a p21 GTPase required for polarity establishment and bud emergence. This finding coupled with recent experiments with mammalian and yeast cells indicating that Cdc42p can interact with and activate Ste20p, a protein kinase that operates in the pheromone pathway, leads us to suggest that Rga1p controls the activity of Cdc42p, which in turn controls the magnitude of signaling in the pheromone pathway via Ste20p.

Amino Acid Sequence↗
PubMed1993

Cell division inhibitors SulA and MinCD prevent formation of the FtsZ ring.

Immunoelectron microscopy was used to assess the effects of inhibitors of cell division on formation of the FtsZ ring in Escherichia coli. Induction of the cell division inhibitor SulA, a component of the SOS response, or the inhibitor MinCD, a component of the min system, blocked formation of the FtsZ ring and led to filamentation. Reversal of SulA inhibition by blocking protein synthesis in SulA-induced filaments led to a resumption of FtsZ ring formation and division. These results suggested that these inhibitors block cell division by preventing FtsZ localization into the ring structure. In addition, analysis of min mutants demonstrated that FtsZ ring formation was also associated with minicell formation, indicating that all septation events in E. coli involve the FtsZ ring.

Adenosine Triphosphatases↗
PubMed1992

Isolation and characterization of ftsZ alleles that affect septal morphology.

The ftsZ gene encodes an essential cell division protein that specifically localizes to the septum of dividing cells. In this study we characterized the effects of the ftsZ2(Rsa) mutation on cell physiology. We found that this mutation caused an altered cell morphology that included minicell formation and an increased average cell length. In addition, this mutation caused a temperature-dependent effect on cell lysis. During this investigation we fortuitously isolated a novel temperature-sensitive ftsZ mutation that consisted of a 6-codon insertion near the 5' end of the gene. This mutation, designated ftsZ26(Ts), caused an altered polar morphology at the permissive temperature and blocked cell division at the nonpermissive temperature. The altered polar morphology resulted from cell division and correlated with an altered geometry of the FtsZ ring. An intragenic cold-sensitive suppressor of ftsZ26(Ts) that caused cell lysis at the nonpermissive temperature was isolated. These results support the hypothesis that the FtsZ ring determines the division site and interacts with the septal biosynthetic machinery.

Alleles↗
PubMed1990

Analysis of ftsZ mutations that confer resistance to the cell division inhibitor SulA (SfiA).

In Escherichia coli, the ftsZ gene is thought to be an essential cell division gene. Several dominant mutations that make lon mutant cells refractory to the cell division inhibitor SulA, sulB9, sulB25, and sfiB114, have been mapped to the ftsZ gene. DNA sequence analysis of these mutations and the sfiB103 mutation confirmed that all of these mutations mapped within the ftsZ gene and revealed that the two sulB mutations were identical and by selection for resistance to higher levels of SulA, contained a second mutation within the ftsZ gene. We therefore propose that these mutations be redesignated ftsZ(Rsa) for resistance to SulA. A procedure involving mutagenesis of ftsZ cloned on low-copy-number vectors was used to isolate three additional ftsZ(Rsa) mutations. DNA sequence analysis of these mutations revealed that they were distinct from the previously isolated mutations. One of these mutations, ftsZ3(Rsa), led to an altered FtsZ protein that could no longer support cell growth but still conferred the Rsa phenotype in the presence of ftsZ+. In addition to being resistant to SulA, all ftsZ(Rsa) mutations also conferred resistance to a LacZ-FtsZ hybrid protein (ZZ). One possibility is that FtsZ functions as a multimer and that FtsZ(Rsa) mutant proteins have an increased ability for multimerization, making them resistant to SulA and ZZ.

Alleles↗
PubMed1990

Interaction between the min locus and ftsZ.

In Escherichia coli, distinct but similar minicell phenotypes resulting from mutation at the minB locus and increased expression of ftsZ suggested a possible interaction between these genes. A four- to fivefold increase in FtsZ resulting from increased gene dosage was found to suppress the lethality of minCD expressed from the lac promoter. Since increased MinCD did not affect the level of FtsZ, this suggested that MinCD may antagonize FtsZ to inhibit its cell division activity. This possibility was supported by the finding that alleles of ftsZ isolated as resistant to the cell division inhibitor SulA were also resistant to MinCD. Among the ftsZ(Rsa) alleles, two appeared to be completely resistant to MinCD as demonstrated by the lack of an effect of MinCD on cell length and a minicell phenotype observed in the absence of a significant increase in FtsZ. It was shown that SulA inhibits cell division independently of MinCD.

Escherichia coli↗