Bni1p Regulates Microtubule-Dependent Nuclear Migration through the Actin Cytoskeleton in Saccharomyces cerevisiae

ABSTRACT The RHO1 gene encodes a yeast homolog of the mammalian RhoA protein. Rho1p is localized to the growth sites and is required for bud formation. We have recently shown that Bni1p is one of the potential downstream target molecules of Rho1p. The BNI1gene is implicated in cytokinesis and the establishment of cell polarity in Saccharomyces cerevisiae but is not essential for cell viability. In this study, we screened for mutations that were synthetically lethal in combination with a bni1 mutation and isolated two genes. They were the previously identifiedPAC1 and NIP100 genes, both of which are implicated in nuclear migration in S. cerevisiae. Pac1p is a homolog of human LIS1, which is required for brain development, whereas Nip100p is a homolog of rat p150Glued, a component of the dynein-activated dynactin complex. Disruption ofBNI1 in either the pac1 or nip100mutant resulted in an enhanced defect in nuclear migration, leading to the formation of binucleate mother cells. The arp1 bni1mutant showed a synthetic lethal phenotype while the cin8 bni1 mutant did not, suggesting that Bni1p functions in a kinesin pathway but not in the dynein pathway. Cells of the pac1 bni1 and nip100 bni1 mutants exhibited a random distribution of cortical actin patches. Cells of the pac1 act1-4 mutant showed temperature-sensitive growth and a nuclear migration defect. These results indicate that Bni1p regulates microtubule-dependent nuclear migration through the actin cytoskeleton. Bni1p lacking the Rho-binding region did not suppress the pac1 bni1 growth defect, suggesting a requirement for the Rho1p-Bni1p interaction in microtubule function.

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Sec3p is involved in secretion and morphogenesis in Saccharomyces cerevisiae.

Molecular Biology of the Cell ◽

10.1091/mbc.8.4.647 ◽

1997 ◽

Vol 8 (4) ◽

pp. 647-662 ◽

Cited By ~ 68

Author(s):

F P Finger ◽

P Novick

Keyword(s):

Saccharomyces Cerevisiae ◽

Temperature Sensitive ◽

Synthetic Lethal ◽

Growth Defects ◽

Actin Structure ◽

Synthetically Lethal ◽

Bud Site ◽

Slow Growing ◽

Late Stages ◽

Synthetic Growth

Two new temperature-sensitive alleles of SEC3, 1 of 10 late-acting SEC genes required for targeting or fusion of post-Golgi secretory vesicles to the plasma membrane in Saccharomyces cerevisiae, were isolated in a screen for temperature-sensitive secretory mutants that are synthetically lethal with sec4-8. The new sec3 alleles affect early as well as late stages of secretion. Cloning and sequencing of the SEC3 gene revealed that it is identical to profilin synthetic lethal 1 (PSL1). The SEC3 gene is not essential because cells depleted of Sec3p are viable although slow growing and temperature sensitive. All of the sec3 alleles genetically interact with a profilin mutation, pfy1-111. The SEC3 gene in high copy suppresses pfy1-111 and sec5-24 and causes synthetic growth defects with ypt1, sec8-9, sec10-2, and sec15-1. Actin structure is only perturbed in conditions of chronic loss of Sec3p function, implying that Sec3p does not directly regulate actin. All alleles of sec3 cause bud site selection defects in homozygous diploids, as do sec4-8 and sec9-4. This suggests that SEC gene products are involved in determining the bud site and is consistent with a role for Sec3p in determining the correct site of exocytosis.

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SRO9, a Multicopy Suppressor of the Bud Gpowth Defect in the Saccharomyces cerevisiae rho3-Deficient Cells, Shows Strong Genetic Interactions With Tropomyosin Genes, Suggesting Its Role in Organization of the Actin Cytoskeleton

Genetics ◽

10.1093/genetics/147.3.1003 ◽

1997 ◽

Vol 147 (3) ◽

pp. 1003-1016

Author(s):

Mitsuhiro Kagami ◽

Akio Toh-e ◽

Yasushi Matsui

Keyword(s):

Saccharomyces Cerevisiae ◽

Actin Cytoskeleton ◽

Actin Filaments ◽

Rna Binding ◽

Small Gtpase ◽

Growth Defect ◽

Cortical Actin ◽

Cytoplasmic Factor ◽

Multicopy Suppressor ◽

Actin Cables

RHO3 encodes a Rho-type small GTPase in the yeast Saccharomyces cerevisiae and is involved in the proper organization of the actin cytoskeleton required for bud growth. SRO9 (YCL37c) was isolated as a multicopy suppressor of a rho3Δ mutation. An Sro9p domain required for function is similar to a domain in the La protein (an RNA-binding protein). Disruption of SRO9 did not affect vegetative growth, even with the simultaneous disruption of an SRO9 homologue, SRO99. However, sro9Δ was synthetically lethal with a disruption of TPM1, which encodes tropomyosin; sro9Δ tpm1Δ cells did not distribute cortical actin patches properly and lysed. We isolated TPM2, the other gene for tropomyosin, as a multicopy suppressor of a tpm1Δ sro9Δ double mutant. Genetic analysis suggests that TPM2 is functionally related to TPM1 and that tropomyosin is important but not essential for cell growth. Overexpression of SRO9 suppressed the growth defect in tpm1Δ tpm2Δ cells, disappearance of cables of actin filaments in both rho3Δ cells and tpm1Δ cells, and temperature sensitivity of actin mutant cells (act1-1 cells), suggesting that Sro9p has a function that overlaps or is related to tropomyosin function. Unlike tropomyosin, Sro9p does not colocalize with actin cables but is diffusely cytoplasmic. These results suggest that Sro9p is a new cytoplasmic factor involved in the organization of actin filaments.

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A Synthetic Lethal Screen Identifies a Role for the Cortical Actin Patch/Endocytosis Complex in the Response to Nutrient Deprivation in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/166.2.707 ◽

2004 ◽

Vol 166 (2) ◽

pp. 707-719 ◽

Cited By ~ 1

Author(s):

Alison Care ◽

Katherine A Vousden ◽

Katie M Binley ◽

Pippa Radcliffe ◽

Janet Trevethick ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Actin Cytoskeleton ◽

Nutrient Limitation ◽

Fluid Phase ◽

Nutrient Deprivation ◽

Cortical Actin ◽

Synthetic Lethal ◽

Fluid Phase Endocytosis ◽

Actin Patch ◽

Proper Response

Abstract Saccharomyces cerevisiae whi2Δ cells are unable to halt cell division in response to nutrient limitation and are sensitive to a wide variety of stresses. A synthetic lethal screen resulted in the isolation of siw mutants that had a phenotype similar to that of whi2Δ. Among these were mutations affecting SIW14, FEN2, SLT2, and THR4. Fluid-phase endocytosis is severely reduced or abolished in whi2Δ, siw14Δ, fen2Δ, and thr4Δ mutants. Furthermore, whi2Δ and siw14Δ mutants produce large actin clumps in stationary phase similar to those seen in prk1Δ ark1Δ mutants defective in protein kinases that regulate the actin cytoskeleton. Overexpression of SIW14 in a prk1Δ strain resulted in a loss of cortical actin patches and cables and was lethal. Overexpression of SIW14 also rescued the caffeine sensitivity of the slt2 mutant isolated in the screen, but this was not due to alteration of the phosphorylation state of Slt2. These observations suggest that endocytosis and the organization of the actin cytoskeleton are required for the proper response to nutrient limitation. This hypothesis is supported by the observation that rvs161Δ, sla1Δ, sla2Δ, vrp1Δ, ypt51Δ, ypt52Δ, and end3Δ mutations, which disrupt the organization of the actin cytoskeleton and/or reduce endocytosis, have a phenotype similar to that of whi2Δ mutants.

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Mutations in the Saccharomyces cerevisiae Type 2A Protein Phosphatase Catalytic Subunit Reveal Roles in Cell Wall Integrity, Actin Cytoskeleton Organization and Mitosis

Genetics ◽

10.1093/genetics/145.2.227 ◽

1997 ◽

Vol 145 (2) ◽

pp. 227-241 ◽

Cited By ~ 4

Author(s):

David R H Evans ◽

Michael J R Stark

Keyword(s):

Saccharomyces Cerevisiae ◽

Actin Cytoskeleton ◽

Protein Phosphatase ◽

Catalytic Subunit ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Cell Integrity ◽

Cortical Actin ◽

Prolonged Incubation ◽

Mutant Cells

Temperature-sensitive mutations were generated in the Saccharomyces cerevisiae PPH22 gene that, together with its homologue PPH21, encode the catalytic subunit of type 2A protein phosphatase (PP2A). At the restrictive temperature (37°), cells dependent solely on pph22ts alleles for PP2A function displayed a rapid arrest of proliferation. Ts– pph22 mutant cells underwent lysis at 37°, showing an accompanying viability loss that was suppressed by inclusion of 1 m sorbitol in the growth medium. Ts– pph22 mutant cells also displayed defects in bud morphogenesis and polarization of the cortical actin cytoskeleton at 37°. PP2A is therefore required for maintenance of cell integrity and polarized growth. On transfer from 24° to 37°, Ts– pph22 mutant cells accumulated a 2N DNA content indicating a cell cycle block before completion of mitosis. However, during prolonged incubation at 37°, many Ts– pph22 mutant cells progressed through an aberrant nuclear division and accumulated multiple nuclei. Ts– pph22 mutant cells also accumulated aberrant microtubule structures at 37°, while under semi-permissive conditions they were sensitive to the microtubule-destabilizing agent benomyl, suggesting that PP2A is required for normal microtubule function. Remarkably, the multiple defects of Ts– pph22 mutant cells were suppressed by a viable allele (SSD1-v1) of the polymorphic SSD1 gene.

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Mutations that enhance the cap2 null mutant phenotype in Saccharomyces cerevisiae affect the actin cytoskeleton, morphogenesis and pattern of growth.

Genetics ◽

10.1093/genetics/135.3.693 ◽

1993 ◽

Vol 135 (3) ◽

pp. 693-709 ◽

Cited By ~ 1

Author(s):

T S Karpova ◽

M M Lepetit ◽

J A Cooper

Keyword(s):

Saccharomyces Cerevisiae ◽

Actin Cytoskeleton ◽

Synthetic Lethality ◽

Actin Binding ◽

Cortical Actin ◽

Gene Encoding ◽

Synthetic Lethal ◽

Capping Protein ◽

Colony Color ◽

Actin Cables

Abstract Mutations conferring synthetic lethality in combination with null mutations in CAP2, the gene encoding the beta subunit of capping protein of Saccharomyces cerevisiae, were obtained in a colony color assay. Monogenic inheritance was found for four mutations, which were attributed to three genetic loci. One mutation, sac6-69, is in the gene encoding fimbrin, another actin-binding protein, which was expected because null mutations in SAC6 and CAP2 are known to be synthetic-lethal. The other two loci were designated slc for synthetic lethality with cap2. These loci include the mutations slc1-66, slc1-87 and slc2-107. The slc mutations are semi-dominant, as shown by incomplete complementation in slc/SLC cap2/cap2 heterozygotes. The slc mutations and sac6-69 interact with each other, as shown by enhanced phenotypes in diheterozygotes. Moreover, the haploid slc2-107 sac6-69 double mutant is inviable. In a CAP2 background, the slc mutations lead to temperature and osmotic sensitivity. They alter the distribution of the actin cytoskeleton, including deficits in the presence of actin cables and the polarization of cortical actin patches. The slc mutations also lead to a pseudomycelial growth pattern. Together these results suggest that slc1 and slc2 encode components of the actin cytoskeleton in yeast and that the actin cytoskeleton can regulate the patterns of growth.

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Saccharomyces cerevisiae SMT4 Encodes an Evolutionarily Conserved Protease With a Role in Chromosome Condensation Regulation

Genetics ◽

10.1093/genetics/158.1.95 ◽

2001 ◽

Vol 158 (1) ◽

pp. 95-107 ◽

Cited By ~ 1

Author(s):

Alexander V Strunnikov ◽

L Aravind ◽

Eugene V Koonin

Keyword(s):

Saccharomyces Cerevisiae ◽

Active Site ◽

Growth Defect ◽

Temperature Sensitive ◽

Site Specific ◽

Active Site Cysteine ◽

Condensin Complex ◽

Evolutionarily Conserved ◽

Synthetically Lethal

Abstract In a search for regulatory genes affecting the targeting of the condensin complex to chromatin in Saccharomyces cerevisiae, we identified a member of the adenovirus protease family, SMT4. SMT4 overexpression suppresses the temperature-sensitive conditional lethal phenotype of smc2-6, but not smc2-8 or smc4-1. A disruption allele of SMT4 has a prominent chromosome phenotype: impaired targeting of Smc4p-GFP to rDNA chromatin. Site-specific mutagenesis of the predicted protease active site cysteine and histidine residues of Smt4p abolishes the SMT4 function in vivo. The previously uncharacterized SIZ1 (SAP and Miz) gene, which encodes a protein containing a predicted DNA-binding SAP module and a Miz finger, is identified as a bypass suppressor of the growth defect associated with the SMT4 disruption. The SIZ1 gene disruption is synthetically lethal with the SIZ2 deletion. We propose that SMT4, SIZ1, and SIZ2 are involved in a novel pathway of chromosome maintenance.

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Suppressors of a Saccharomyces cerevisiae pkc1 mutation identify alleles of the phosphatase gene PTC1 and of a novel gene encoding a putative basic leucine zipper protein.

Genetics ◽

10.1093/genetics/141.4.1275 ◽

1995 ◽

Vol 141 (4) ◽

pp. 1275-1285 ◽

Cited By ~ 13

Author(s):

K N Huang ◽

L S Symington

Keyword(s):

Protein Kinase ◽

Leucine Zipper ◽

Mapk Pathway ◽

Mitogen Activated Protein Kinase ◽

Growth Defect ◽

Temperature Sensitive ◽

Gene Encoding ◽

Basic Leucine Zipper ◽

Mapk Cascades ◽

Synthetically Lethal

Abstract The PKC1 gene product, protein kinase C, regulates a mitogen-activated protein kinase (MAPK) cascade, which is implicated in cell wall metabolism. Previously, we identified the pkc1-4 allele in a screen for mutants with increased rates of recombination, indicating that PKC1 may also regulate DNA metabolism. The pkc1-4 allele also conferred a temperature-sensitive (ts) growth defect. Extragenic suppressors were isolated that suppress both the ts and hyperrecombination phenotypes conferred by the pkc1-4 mutation. Eight of these suppressors for into two complementation groups, designated KCS1 and KCS2. KCS1 was cloned and found to encode a novel protein with homology to the basic leucine zipper family of transcription factors. KCS2 is allelic with PTC1, a previously identified type 2C serine/threonine protein phosphatase. Although mutation of either KCS1 or PTC1 causes little apparent phenotype, the kcs1 delta ptc1 delta double mutant fails to grow at 30 degrees. Furthermore, the ptc1 deletion mutation is synthetically lethal in combination with a mutation in MPK1, which encodes a MAPK homologue proposed to act in the PKC1 pathway. Because PTC1 was initially isolated as a component of the Hog1p MAPK pathway, it appears that these two MAPK cascades share a common regulatory feature.

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The Putative RNA Helicase Dbp6p Functionally Interacts With Rpl3p, Nop8p and the Novel trans-acting Factor Rsa3p During Biogenesis of 60S Ribosomal Subunits in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/166.4.1687 ◽

2004 ◽

Vol 166 (4) ◽

pp. 1687-1699

Author(s):

Jesús de la Cruz ◽

Thierry Lacombe ◽

Olivier Deloche ◽

Patrick Linder ◽

Dieter Kressler

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribosome Biogenesis ◽

Null Allele ◽

Ribosomal Subunit ◽

Interaction Network ◽

The Novel ◽

Rna Helicases ◽

Ribosomal Subunits ◽

Synthetic Lethal ◽

Synthetically Lethal

Abstract Ribosome biogenesis requires at least 18 putative ATP-dependent RNA helicases in Saccharomyces cerevisiae. To explore the functional environment of one of these putative RNA helicases, Dbp6p, we have performed a synthetic lethal screen with dbp6 alleles. We have previously characterized the nonessential Rsa1p, whose null allele is synthetically lethal with dbp6 alleles. Here, we report on the characterization of the four remaining synthetic lethal mutants, which reveals that Dbp6p also functionally interacts with Rpl3p, Nop8p, and the so-far-uncharacterized Rsa3p (ribosome assembly 3). The nonessential Rsa3p is a predominantly nucleolar protein required for optimal biogenesis of 60S ribosomal subunits. Both Dbp6p and Rsa3p are associated with complexes that most likely correspond to early pre-60S ribosomal particles. Moreover, Rsa3p is co-immunoprecipitated with protA-tagged Dbp6p under low salt conditions. In addition, we have established a synthetic interaction network among factors involved in different aspects of 60S-ribosomal-subunit biogenesis. This extensive genetic analysis reveals that the rsa3 null mutant displays some specificity by being synthetically lethal with dbp6 alleles and by showing some synthetic enhancement with the nop8-101 and the rsa1 null allele.

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A Role for the Actin Cytoskeleton of Saccharomyces cerevisiae in Bipolar Bud-Site Selection

The Journal of Cell Biology ◽

10.1083/jcb.136.1.111 ◽

1997 ◽

Vol 136 (1) ◽

pp. 111-123 ◽

Cited By ~ 58

Author(s):

Shirley Yang ◽

Kathryn R. Ayscough ◽

David G. Drubin

Keyword(s):

Saccharomyces Cerevisiae ◽

Actin Cytoskeleton ◽

Site Selection ◽

Mitotic Checkpoint ◽

Cell Cortex ◽

Spatial Cues ◽

Genes Encoding ◽

Mother And Daughter ◽

Mother Cells ◽

Bud Site

Saccharomyces cerevisiae cells select bud sites according to one of two predetermined patterns. MATa and MATα cells bud in an axial pattern, and MATa/α cells bud in a bipolar pattern. These budding patterns are thought to depend on the placement of spatial cues at specific sites in the cell cortex. Because cytoskeletal elements play a role in organizing the cytoplasm and establishing distinct plasma membrane domains, they are well suited for positioning bud-site selection cues. Indeed, the septin-containing neck filaments are crucial for establishing the axial budding pattern characteristic of MATa and MATα cells. In this study, we determined the budding patterns of cells carrying mutations in the actin gene or in genes encoding actin-associated proteins: MATa/α cells were defective in the bipolar budding pattern, but MATa and MATα cells still exhibit a normal axial budding pattern. We also observed that MATa/α actin cytoskeleton mutant daughter cells correctly position their first bud at the distal pole of the cell, but mother cells position their buds randomly. The actin cytoskeleton therefore functions in generation of the bipolar budding pattern and is required specifically for proper selection of bud sites in mother MATa/α cells. These observations and the results of double mutant studies support the conclusion that different rules govern bud-site selection in mother and daughter MATa/α cells. A defective bipolar budding pattern did not preclude an sla2-6 mutant from undergoing pseudohyphal growth, highlighting the central role of daughter cell bud-site selection cues in the formation of pseudohyphae. Finally, by examining the budding patterns of mad2-1 mitotic checkpoint mutants treated with benomyl to depolymerize their microtubules, we confirmed and extended previous evidence indicating that microtubules do not function in axial or bipolar bud-site selection.

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PTA1, an essential gene of Saccharomyces cerevisiae affecting pre-tRNA processing.

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.3843 ◽

1992 ◽

Vol 12 (9) ◽

pp. 3843-3856 ◽

Cited By ~ 20

Author(s):

J P O'Connor ◽

C L Peebles

Keyword(s):

Saccharomyces Cerevisiae ◽

Gene Product ◽

Genomic Library ◽

Essential Gene ◽

Growth Defect ◽

Temperature Sensitive ◽

Putative Open Reading Frame ◽

Reading Frame ◽

Trna Processing ◽

Chromosome I

We have identified an essential Saccharomyces cerevisiae gene, PTA1, that affects pre-tRNA processing. PTA1 was initially defined by a UV-induced mutation, pta1-1, that causes the accumulation of all 10 end-trimmed, intron-containing pre-tRNAs and temperature-sensitive but osmotic-remedial growth. pta1-1 does not appear to be an allele of any other known gene affecting pre-tRNA processing. Extracts prepared from pta1-1 strains had normal pre-tRNA splicing endonuclease activity. pta1-1 was suppressed by the ochre suppressor tRNA gene SUP11, indicating that the pta1-1 mutation creates a termination codon within a protein reading frame. The PTA1 gene was isolated from a genomic library by complementation of the pta1-1 growth defect. Episome-borne PTA1 directs recombination to the pta1-1 locus. PTA1 has been mapped to the left arm of chromosome I near CDC24; the gene was sequenced and could encode a protein of 785 amino acids with a molecular weight of 88,417. No other protein sequences similar to that of the predicted PTA1 gene product have been identified within the EMBL or GenBank data base. Disruption of PTA1 near the carboxy terminus of the putative open reading frame was lethal. Possible functions of the PTA1 gene product are discussed.

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