scholarly journals Identification and nuclear localization of yeast pre-messenger RNA processing components: RNA2 and RNA3 proteins.

1986 ◽  
Vol 103 (6) ◽  
pp. 2103-2112 ◽  
Author(s):  
R L Last ◽  
J L Woolford
Keyword(s):  
Nuclear Localization ◽  
Indirect Immunofluorescence ◽  
Rna Processing ◽  
Copy Number ◽  
Messenger Rna ◽  
Subcellular Fractionation ◽  
Yeast Cells ◽  
High Copy Number ◽  
Temperature Sensitive

Temperature-sensitive mutations in the RNA2 through RNA11 genes of yeast prevent the processing of nuclear pre-mRNAs. We have raised antisera that detect the RNA2 and RNA3 proteins in immunoblots of extracts of yeast containing high copy number RNA2 and RNA3 plasmids. Subcellular fractionation of yeast cells that overproduce the RNA2 and RNA3 proteins has revealed that these proteins are enriched in nuclear fractions. Indirect immunofluorescence results have indicated that these proteins are localized in yeast nuclei. These localization results are consistent with the fact that these genes have a role in processing yeast pre-mRNA.

Genetic interactions between CDC31 and KAR1, two genes required for duplication of the microtubule organizing center in Saccharomyces cerevisiae.

Genetics ◽  
1994 ◽  
Vol 137 (2) ◽  
pp. 407-422 ◽  
Author(s):  
E A Vallen ◽  
W Ho ◽  
M Winey ◽  
M D Rose
Keyword(s):  
Copy Number ◽  
Gene Dosage ◽  
Spindle Pole Body ◽  
Direct Interaction ◽  
Spindle Pole ◽  
High Copy Number ◽  
Temperature Sensitive ◽  
Microtubule Organizing Center ◽  
Recessive Mutations ◽  
Organizing Center

Abstract KAR1 encodes an essential component of the yeast spindle pole body (SPB) that is required for karyogamy and SPB duplication. A temperature-sensitive mutation, kar1-delta 17, mapped to a region required for SPB duplication and for localization to the SPB. To identify interacting SPB proteins, we isolated 13 dominant mutations and 3 high copy number plasmids that suppressed the temperature sensitivity of kar1-delta 17. Eleven extragenic suppressor mutations mapped to two linkage groups, DSK1 and DSK2. The extragenic suppressors were specific for SPB duplication and did not suppress karyogamy-defective alleles. The major class, DSK1, consisted of mutations in CDC31. CDC31 is required for SPB duplication and encodes a calmodulin-like protein that is most closely related to caltractin/centrin, a protein associated with the Chlamydomonas basal body. The high copy number suppressor plasmids contained the wild-type CDC31 gene. One CDC31 suppressor allele conferred a temperature-sensitive defect in SPB duplication, which was counter-suppressed by recessive mutations in KAR1. In spite of the evidence for a direct interaction, the strongest CDC31 alleles, as well as both DSK2 alleles, suppressed a complete deletion of KAR1. However, the CDC31 alleles also made the cell supersensitive to KAR1 gene dosage, arguing against a simple bypass mechanism of suppression. We propose a model in which Kar1p helps localize Cdc31p to the SPB and that Cdc31p then initiates SPB duplication via interaction with a downstream effector.

scholarly journals Cellular localization of RNA14p and RNA15p, two yeast proteins involved in mRNA stability

1994 ◽  
Vol 107 (4) ◽  
pp. 913-921
Author(s):  
N. Bonneaud ◽  
L. Minvielle-Sebastia ◽  
C. Cullin ◽  
F. Lacroute
Keyword(s):  
Nuclear Localization ◽  
Mrna Stability ◽  
Cellular Localization ◽  
Tail Length ◽  
Genetic Data ◽  
Subcellular Fractionation ◽  
Rapid Decrease ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Mrna Levels

RNA14 and RNA15 were originally identified by temperature-sensitive mutations that cause a rapid decrease in poly(A)-tail length and overall mRNA levels at the restrictive temperature. We have raised antibodies to the RNA14 and RNA15 proteins, and used subcellular fractionation and immunofluorescence to localize these proteins within the yeast cell. RNA14p is a 73 kDa protein found in both the nucleus and the cytoplasm, whilst RNA15p is a 42 kDa protein detected only in the nucleus. The observation that both proteins are found in the nucleus is in agreement with previous genetic data which suggest an interaction between RNA14p and RNA15p. Also the joint nuclear localization is consistent with the biochemical data suggesting a role in polyadenylation. The detection of significant amounts of RNA14p in the cytoplasm opens the possibility of a second function for this protein, either in cytoplasmic regulation of mRNA deadenylation or, more interestingly, in mRNA stability.

scholarly journals Nuclear and mitochondrial inheritance in yeast depends on novel cytoplasmic structures defined by the MDM1 protein.

1992 ◽  
Vol 118 (2) ◽  
pp. 385-395 ◽  
Author(s):  
S J McConnell ◽  
M P Yaffe
Keyword(s):  
Indirect Immunofluorescence ◽  
Protein Product ◽  
Cellular Level ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Animal Cells ◽  
Keratin Gene ◽  
Reading Frame ◽  
Cytoplasmic Structures

The mdml mutation causes temperature-sensitive growth and defective transfer of nuclei and mitochondria into developing buds of yeast cells at the nonpermissive temperature. The MDM1 gene was cloned by complementation, and its sequence revealed an open reading frame encoding a potential protein product of 51.5 kD. This protein displays amino acid sequence similarities to hamster vimentin and mouse epidermal keratin. Gene disruption demonstrated that MDM1 is essential for mitotic growth. Antibodies against the MDM1 protein recognized a 51-kD polypeptide that was localized by indirect immunofluorescence to a novel pattern of spots and punctate arrays distributed throughout the yeast cell cytoplasm. These structures disappeared after shifting mdm1 mutant cells to the nonpermissive temperature, although the cellular level of MDM1 protein was unchanged. Affinity-purified antibodies against MDM1 also specifically recognized intermediate filaments by indirect immunofluorescence of animal cells. These results suggest that novel cytoplasmic structures containing the MDM1 protein mediate organelle inheritance in yeast.

MDS1, a dosage suppressor of an mck1 mutant, encodes a putative yeast homolog of glycogen synthase kinase 3

1994 ◽  
Vol 14 (1) ◽  
pp. 831-839
Author(s):  
J W Puziss ◽  
T A Hardy ◽  
R B Johnson ◽  
P J Roach ◽  
P Hieter
Keyword(s):  
Protein Kinase ◽  
Copy Number ◽  
Glycogen Synthase ◽  
Glycogen Synthase Kinase ◽  
High Copy Number ◽  
Glycogen Synthase Kinase 3 ◽  
Temperature Sensitive ◽  
Protein Kinase Activity ◽  
Nucleotide Sequence Data ◽  
Cold Sensitive

The yeast gene MCK1 encodes a serine/threonine protein kinase that is thought to function in regulating kinetochore activity and entry into meiosis. Disruption of MCK1 confers a cold-sensitive phenotype, a temperature-sensitive phenotype, and sensitivity to the microtubule-destabilizing drug benomyl and leads to loss of chromosomes during growth on benomyl. A dosage suppression selection was used to identify genes that, when present at high copy number, could suppress the cold-sensitive phenotype of mck1::HIS3 mutant cells. Several unique classes of clones were identified, and one of these, designated MDS1, has been characterized in some detail. Nucleotide sequence data reveal that MDS1 encodes a serine/threonine protein kinase that is highly homologous to the shaggy/zw3 kinase in Drosophila melanogaster and its functional homolog, glycogen synthase kinase 3, in rats. The presence of MDS1 in high copy number rescues both the cold-sensitive and the temperature-sensitive phenotypes, but not the benomyl-sensitive phenotype, associated with the disruption of MCK1. Analysis of strains harboring an mds1 null mutation demonstrates that MDS1 is not essential during normal vegetative growth but appears to be required for meiosis. Finally, in vitro experiments indicate that the proteins encoded by both MCK1 and MDS1 possess protein kinase activity with substrate specificity similar to that of mammalian glycogen synthase kinase 3.

scholarly journals MDS1, a dosage suppressor of an mck1 mutant, encodes a putative yeast homolog of glycogen synthase kinase 3.

1994 ◽  
Vol 14 (1) ◽  
pp. 831-839 ◽  
Author(s):  
J W Puziss ◽  
T A Hardy ◽  
R B Johnson ◽  
P J Roach ◽  
P Hieter
Keyword(s):  
Protein Kinase ◽  
Copy Number ◽  
Glycogen Synthase ◽  
Glycogen Synthase Kinase ◽  
High Copy Number ◽  
Glycogen Synthase Kinase 3 ◽  
Temperature Sensitive ◽  
Protein Kinase Activity ◽  
Nucleotide Sequence Data ◽  
Cold Sensitive

The yeast gene MCK1 encodes a serine/threonine protein kinase that is thought to function in regulating kinetochore activity and entry into meiosis. Disruption of MCK1 confers a cold-sensitive phenotype, a temperature-sensitive phenotype, and sensitivity to the microtubule-destabilizing drug benomyl and leads to loss of chromosomes during growth on benomyl. A dosage suppression selection was used to identify genes that, when present at high copy number, could suppress the cold-sensitive phenotype of mck1::HIS3 mutant cells. Several unique classes of clones were identified, and one of these, designated MDS1, has been characterized in some detail. Nucleotide sequence data reveal that MDS1 encodes a serine/threonine protein kinase that is highly homologous to the shaggy/zw3 kinase in Drosophila melanogaster and its functional homolog, glycogen synthase kinase 3, in rats. The presence of MDS1 in high copy number rescues both the cold-sensitive and the temperature-sensitive phenotypes, but not the benomyl-sensitive phenotype, associated with the disruption of MCK1. Analysis of strains harboring an mds1 null mutation demonstrates that MDS1 is not essential during normal vegetative growth but appears to be required for meiosis. Finally, in vitro experiments indicate that the proteins encoded by both MCK1 and MDS1 possess protein kinase activity with substrate specificity similar to that of mammalian glycogen synthase kinase 3.

scholarly journals Suppressors of Bir1p (Survivin) Identify Roles for the Chromosomal Passenger Protein Pic1p (INCENP) and the Replication Initiation Factor Psf2p in Chromosome Segregation

2005 ◽  
Vol 25 (20) ◽  
pp. 9000-9015 ◽  
Author(s):  
Han-Kuei Huang ◽  
Julie M. Bailis ◽  
Joel D. Leverson ◽  
Eliana B. Gómez ◽  
Susan L. Forsburg ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Chromosome Segregation ◽  
Copy Number ◽  
Replication Initiation ◽  
High Copy Number ◽  
Temperature Sensitive ◽  
Chromosome Missegregation ◽  
Chromosomal Passenger ◽  
Sensitive Allele ◽  
Passenger Protein

ABSTRACT Fission yeast Bir1p/Cut17p/Pbh1p, the homolog of human Survivin, is a conserved chromosomal passenger protein that is required for cell division and cytokinesis. To study how Bir1p promotes accurate segregation of chromosomes, we generated and analyzed a temperature-sensitive allele, bir1-46, and carried out genetic screens to find genes that interact with bir1 + . We identified Psf2p, a component of the GINS complex required for DNA replication initiation, as a high-copy-number suppressor of the bir1-46 growth defect. Loss of Psf2p function by depletion or deletion or by use of a temperature-sensitive allele, psf2-209, resulted in chromosome missegregation that was associated with mislocalization of Bir1p. We also found that the human homolog of Psf2p, PSF2, was required for proper chromosome segregation. In addition, we observed that high-copy-number expression of Pic1p, the fission yeast homolog of INCENP (inner centromere protein), suppressed bir1-46. Pic1p exhibited a localization pattern typical of chromosomal passenger proteins. Deletion of pic1 + caused chromosome missegregation phenotypes similar to those of bir1-46. Our data suggest that Bir1p and Pic1p act as part of a conserved chromosomal passenger complex and that Psf2p/GINS indirectly affects the localization and function of this complex in chromosome segregation, perhaps through an S-phase role in centromere replication.

scholarly journals Yeast mitochondria contain ATP-sensitive, reversible actin-binding activity.

1994 ◽  
Vol 5 (7) ◽  
pp. 807-818 ◽  
Author(s):  
D A Lazzarino ◽  
I Boldogh ◽  
M G Smith ◽  
J Rosand ◽  
L A Pon
Keyword(s):  
Outer Membrane Proteins ◽  
Subcellular Fractionation ◽  
Binding Activity ◽  
Yeast Cells ◽  
Actin Binding ◽  
Mitochondrial Outer Membrane ◽  
Temperature Sensitive ◽  
Yeast Mitochondria ◽  
Mother Cells

Sedimentation assays were used to demonstrate and characterize binding of isolated yeast mitochondria to phalloidin-stabilized yeast F-actin. These actin-mitochondrial interactions are ATP sensitive, saturable, reversible, and do not depend upon mitochondrial membrane potential. Protease digestion of mitochondrial outer membrane proteins or saturation of myosin-binding sites on F-actin with the S1 subfragment of skeletal myosin block binding. These observations indicate that a protein (or proteins) on the mitochondrial surface mediates ATP-sensitive, reversible binding of mitochondria to the lateral surface of microfilaments. Actin copurifies with mitochondria during subcellular fractionation and is released from the organelle upon treatment with ATP. Thus, actin-mitochondrial interactions resembling those observed in vitro may also exist in intact yeast cells. Finally, a yeast mutant bearing a temperature-sensitive mutation in the actin-encoding ACT1 gene (act1-3) displays temperature-dependent defects in transfer of mitochondria from mother cells to newly developed buds during yeast cell mitosis.

scholarly journals The Tail of a Yeast Class V Myosin, Myo2p, Functions as a Localization Domain

1999 ◽  
Vol 10 (4) ◽  
pp. 1001-1017 ◽  
Author(s):  
Samara L. Reck-Peterson ◽  
Peter J. Novick ◽  
Mark S. Mooseker
Keyword(s):  
Membrane Trafficking ◽  
Subcellular Fractionation ◽  
Dominant Negative ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Tail Domain ◽  
Class V ◽  
Gal1 Promoter ◽  
Fractionation Pattern ◽  
Sensitive Allele

Myo2p is a yeast class V myosin that functions in membrane trafficking. To investigate the function of the carboxyl-terminal-tail domain of Myo2p, we have overexpressed this domain behind the regulatable GAL1 promoter (MYO2DN). Overexpression of the tail domain of Myo2p results in a dominant–negative phenotype that is phenotypically similar to a temperature-sensitive allele of myo2, myo2–66. The tail domain of Myo2p is sufficient for localization at low- expression levels and causes mislocalization of the endogenous Myo2p from sites of polarized cell growth. Subcellular fractionation of polarized, mechanically lysed yeast cells reveals that Myo2p is present predominantly in a 100,000 × g pellet. The Myo2p in this pellet is not solubilized by Mg++-ATP or Triton X-100, but is solubilized by high salt. Tail overexpression does not disrupt this fractionation pattern, nor do mutations in sec4, sec3, sec9, cdc42, or myo2. These results show that overexpression of the tail domain of Myo2p does not compete with the endogenous Myo2p for assembly into a pelletable structure, but does compete with the endogenous Myo2p for a factor that is necessary for localization to the bud tip.

scholarly journals The Rho-GEF Rom2p Localizes to Sites of Polarized Cell Growth and Participates in Cytoskeletal Functions inSaccharomyces cerevisiae

1997 ◽  
Vol 8 (10) ◽  
pp. 1829-1844 ◽  
Author(s):  
Brendan D. Manning ◽  
Ramesh Padmanabha ◽  
Michael Snyder
Keyword(s):  
Cell Growth ◽  
Copy Number ◽  
High Copy Number ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Rho Proteins ◽  
Growth Defects ◽  
Polarized Cell Growth ◽  
Morphological Defects ◽  
Increased Sensitivity

Rom2p is a GDP/GTP exchange factor for Rho1p and Rho2p GTPases; Rho proteins have been implicated in control of actin cytoskeletal rearrangements. ROM2 and RHO2 were identified in a screen for high-copy number suppressors ofcik1Δ, a mutant defective in microtubule-based processes in Saccharomyces cerevisiae. A Rom2p::3XHA fusion protein localizes to sites of polarized cell growth, including incipient bud sites, tips of small buds, and tips of mating projections. Disruption of ROM2 results in temperature-sensitive growth defects at 11°C and 37°C.rom2Δ cells exhibit morphological defects. At permissive temperatures, rom2Δ cells often form elongated buds and fail to form normal mating projections after exposure to pheromone; at the restrictive temperature, small budded cells accumulate. High-copy number plasmids containing eitherROM2 or RHO2 suppress the temperature-sensitive growth defects of cik1Δ andkar3Δ strains. KAR3 encodes a kinesin-related protein that interacts with Cik1p. Furthermore,rom2Δ strains exhibit increased sensitivity to the microtubule depolymerizing drug benomyl. These results suggest a role for Rom2p in both polarized morphogenesis and functions of the microtubule cytoskeleton.

scholarly journals Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 21-29 ◽  
Author(s):  
David R H Evans ◽  
Brian A Hemmings
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Protein Function ◽  
Mutational Analysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Active Site Residues ◽  
Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

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