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.

Dolichol phosphate mannose synthase is required in vivo for glycosyl phosphatidylinositol membrane anchoring, O mannosylation, and N glycosylation of protein in Saccharomyces cerevisiae

1990 ◽  
Vol 10 (11) ◽  
pp. 5796-5805
Author(s):  
P Orlean
Keyword(s):  
Molecular Size ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
In Vitro Mutagenesis ◽  
Nonpermissive Temperature ◽  
Gpi Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
Covalent Modifications

Glycosyl phosphatidylinositol (GPI) anchoring, N glycosylation, and O mannosylation of protein occur in the rough endoplasmic reticulum and involve transfer of precursor structures that contain mannose. Direct genetic evidence is presented that dolichol phosphate mannose (Dol-P-Man) synthase, which transfers mannose from GDPMan to the polyisoprenoid dolichol phosphate, is required in vivo for all three biosynthetic pathways leading to these covalent modifications of protein in yeast cells. Temperature-sensitive yeast mutants were isolated after in vitro mutagenesis of the yeast DPM1 gene. At the nonpermissive temperature of 37 degrees C, the dpm1 mutants were blocked in [2-3H]myo-inositol incorporation into protein and accumulated a lipid that could be radiolabeled with both [2-3H]myo-inositol and [2-3H]glucosamine and met existing criteria for an intermediate in GPI anchor biosynthesis. The likeliest explanation for these results is that Dol-P-Man donates the mannose residues needed for completion of the GPI anchor precursor lipid before it can be transferred to protein. Dol-P-Man synthase is also required in vivo for N glycosylation of protein, because (i) dpm1 cells were unable to make the full-length precursor Dol-PP-GlcNAc2Man9Glc3 and instead accumulated the intermediate Dol-PP-GlcNAc2Man5 in their pool of lipid-linked precursor oligosaccharides and (ii) truncated, endoglycosidase H-resistant oligosaccharides were transferred to the N-glycosylated protein invertase after a shift to 37 degrees C. Dol-P-Man synthase is also required in vivo for O mannosylation of protein, because chitinase, normally a 150-kDa O-mannosylated protein, showed a molecular size of 60 kDa, the size predicted for the unglycosylated protein, after shift of the dpm1 mutant to the nonpermissive temperature.

scholarly journals The BN51 protein is a polymerase (Pol)-specific subunit of RNA Pol III which reveals a link between Pol III transcription and pre-rRNA processing.

1995 ◽  
Vol 15 (1) ◽  
pp. 94-101 ◽  
Author(s):  
A J Jackson ◽  
M Ittmann ◽  
B F Pugh
Keyword(s):  
Yeast Cells ◽  
Temperature Sensitive ◽  
28S Rrna ◽  
Nonpermissive Temperature ◽  
Biochemical Evidence ◽  
Pol I ◽  
Nuclear Extracts ◽  
A Subunit ◽  
Pol Iii ◽  
Mammalian System

The three eukaryotic nuclear RNA polymerase (Pol) contain common and unique subunits. Cloning of the unique Pol III subunit genes in yeast cells has revealed a potential homolog in the mammalian system, the BN51 gene. The human BN51 gene was originally isolated as a suppressor of a temperature-sensitive cell cycle mutant of BHK cells (tsBN51). Although tsBN51 cells have a marked decrease in RNA Pol III activity at the nonpermissive temperature, direct biochemical evidence for the BN51 protein being a human Pol III subunit was lacking. Using antibodies directed against the BN51 protein, we show the following: (i) the BN51 protein copurifies with Pol III activity, (ii) Pol III activity can be specifically immunoprecipitated from HeLa nuclear extracts, and (iii) the immunopurified BN51 complex is active in restoring both nonspecific and promoter-specific Pol III activity. Our findings provide direct biochemical evidence for BN51 being a Pol III-specific subunit. Despite the fact that BN51 is not a subunit of Pol I, the production of mature Pol I transcripts is inhibited in tsBN51 cells at the nonpermissive temperature. tsBN51 cells appear defective in processing the 32S precursor rRNA into mature 5.8S and 28S rRNA at the nonpermissive temperature. We surmise that ribosome assembly has halted because of the loss of Pol III transcripts. Thus, there is regulation of the synthesis of mature Pol I transcripts by a posttranscriptional mechanism based on the availability of Pol III transcripts.

scholarly journals Mps3p is a novel component of the yeast spindle pole body that interacts with the yeast centrin homologue Cdc31p

2002 ◽  
Vol 159 (6) ◽  
pp. 945-956 ◽  
Author(s):  
Sue L. Jaspersen ◽  
Thomas H. Giddings ◽  
Mark Winey
Keyword(s):  
Mitotic Spindle ◽  
Spindle Pole Body ◽  
Integral Membrane Protein ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Reading Frame ◽  
Pole Body ◽  
Monopolar Spindle

Accurate duplication of the Saccharomyces cerevisiae spindle pole body (SPB) is required for formation of a bipolar mitotic spindle. We identified mutants in SPB assembly by screening a temperature-sensitive collection of yeast for defects in SPB incorporation of a fluorescently marked integral SPB component, Spc42p. One SPB assembly mutant contained a mutation in a previously uncharacterized open reading frame that we call MPS3 (for monopolar spindle). mps3-1 mutants arrest in mitosis with monopolar spindles at the nonpermissive temperature, suggesting a defect in SPB duplication. Execution point experiments revealed that MPS3 function is required for the first step of SPB duplication in G1. Like cells containing mutations in two other genes required for this step of SPB duplication (CDC31 and KAR1), mps3-1 mutants arrest with a single unduplicated SPB that lacks an associated half-bridge. MPS3 encodes an essential integral membrane protein that localizes to the SPB half-bridge. Genetic interactions between MPS3 and CDC31 and binding of Cdc31p to Mps3p in vitro, as well as the fact that Cdc31p localization to the SPB is partially dependent on Mps3p function, suggest that one function for Mps3p during SPB duplication is to recruit Cdc31p, the yeast centrin homologue, to the half-bridge.

scholarly journals Dolichol phosphate mannose synthase is required in vivo for glycosyl phosphatidylinositol membrane anchoring, O mannosylation, and N glycosylation of protein in Saccharomyces cerevisiae.

1990 ◽  
Vol 10 (11) ◽  
pp. 5796-5805 ◽  
Author(s):  
P Orlean
Keyword(s):  
Molecular Size ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
In Vitro Mutagenesis ◽  
Nonpermissive Temperature ◽  
Gpi Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
Covalent Modifications

Glycosyl phosphatidylinositol (GPI) anchoring, N glycosylation, and O mannosylation of protein occur in the rough endoplasmic reticulum and involve transfer of precursor structures that contain mannose. Direct genetic evidence is presented that dolichol phosphate mannose (Dol-P-Man) synthase, which transfers mannose from GDPMan to the polyisoprenoid dolichol phosphate, is required in vivo for all three biosynthetic pathways leading to these covalent modifications of protein in yeast cells. Temperature-sensitive yeast mutants were isolated after in vitro mutagenesis of the yeast DPM1 gene. At the nonpermissive temperature of 37 degrees C, the dpm1 mutants were blocked in [2-3H]myo-inositol incorporation into protein and accumulated a lipid that could be radiolabeled with both [2-3H]myo-inositol and [2-3H]glucosamine and met existing criteria for an intermediate in GPI anchor biosynthesis. The likeliest explanation for these results is that Dol-P-Man donates the mannose residues needed for completion of the GPI anchor precursor lipid before it can be transferred to protein. Dol-P-Man synthase is also required in vivo for N glycosylation of protein, because (i) dpm1 cells were unable to make the full-length precursor Dol-PP-GlcNAc2Man9Glc3 and instead accumulated the intermediate Dol-PP-GlcNAc2Man5 in their pool of lipid-linked precursor oligosaccharides and (ii) truncated, endoglycosidase H-resistant oligosaccharides were transferred to the N-glycosylated protein invertase after a shift to 37 degrees C. Dol-P-Man synthase is also required in vivo for O mannosylation of protein, because chitinase, normally a 150-kDa O-mannosylated protein, showed a molecular size of 60 kDa, the size predicted for the unglycosylated protein, after shift of the dpm1 mutant to the nonpermissive temperature.

scholarly journals Vaccinia Virus Mutants with Alanine Substitutions in the Conserved G5R Gene Fail To Initiate Morphogenesis at the Nonpermissive Temperature

2004 ◽  
Vol 78 (19) ◽  
pp. 10238-10248 ◽  
Author(s):  
Flavio G. da Fonseca ◽  
Andrea S. Weisberg ◽  
Maria F. Caeiro ◽  
Bernard Moss
Keyword(s):  
Dna Replication ◽  
Vaccinia Virus ◽  
Viral Gene Expression ◽  
Proteolytic Cleavage ◽  
Viral Gene ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Reading Frame ◽  
Charged Amino Acids ◽  
Core Proteins

ABSTRACT The initial characterization of the product of the vaccinia virus G5R gene, which is conserved in all poxviruses sequenced to date, is described. The G5 protein was detected in the core fraction of purified virions, and transcription and translation of the G5R open reading frame occurred early in infection, independently of DNA replication. Attempts to delete the G5R gene and isolate a replication-competent virus were unsuccessful, suggesting that G5R encodes an essential function. We engineered vaccinia virus mutants with clusters of charged amino acids changed to alanines and determined that several were unable to replicate at 40°C but grew well at 37°C. At the nonpermissive temperature, viral gene expression and DNA replication and processing were unperturbed. However, tyrosine phosphorylation and proteolytic cleavage of the A17 membrane protein and proteolytic cleavage of core proteins were inhibited at 40°C, suggesting an assembly defect. The cytoplasm of cells that had been infected at the nonpermissive temperature contained large granular areas devoid of cellular organelles or virus structures except for occasional short crescent-shaped membranes and electron-dense lacy structures. The temperature-sensitive phenotype of the G5R mutants closely resembled the phenotypes of vaccinia virus mutants carrying conditionally lethal F10R protein kinase and H5R mutations. F10, although required for phosphorylation of A17 and viral membrane formation, was synthesized by the G5R mutants under nonpermissive conditions. An intriguing possibility is that G5 participates in the formation of viral membranes, a poorly understood event in poxvirus assembly.

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.

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.

scholarly journals Genetic analysis of temperature-sensitive lethal mutants of Salmonella typhimurium.

Genetics ◽  
1989 ◽  
Vol 123 (4) ◽  
pp. 625-633 ◽  
Author(s):  
M B Schmid ◽  
N Kapur ◽  
D R Isaacson ◽  
P Lindroos ◽  
C Sharpe
Keyword(s):  
High Temperature ◽  
Genetic Analysis ◽  
Salmonella Typhimurium ◽  
Complex Medium ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Anaerobic Conditions ◽  
Temperature Growth ◽  
Lethal Phenotype ◽  
Lethal Mutations

Abstract We have isolated 440 mutants of Salmonella typhimurium that show temperature-sensitive growth on complex medium at 44 degrees. Approximately 16% of the mutations in these strains have been mapped to 17 chromosomal locations; two of these chromosomal locations seem to include several essential genes. Genetic analysis of the mutations suggests that the collection saturates the genes readily mutable to a ts lethal phenotype in S. typhimurium. Physiological characteristics of the ts lethal mutants were tested: 6% of the mutants can grow at high temperature under anaerobic conditions, 17% can grow when the medium includes 0.5 M KCl, and 9% of the mutants die after a 2-hr incubation at the nonpermissive temperature. Most ts lethal mutations in this collection probably affect genes required for growth at all temperatures (not merely during high temperature growth) since Tn10 insertions that cause a temperature-sensitive lethal phenotype are rare.

scholarly journals p50Cdc37 Can Buffer the Temperature-Sensitive Properties of a Mutant of Hck

2000 ◽  
Vol 20 (18) ◽  
pp. 6984-6995 ◽  
Author(s):  
Glen Scholz ◽  
Steven D. Hartson ◽  
Kellie Cartledge ◽  
Nathan Hall ◽  
Jieya Shao ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Active Form ◽  
Tumor Development ◽  
Kinase Domain ◽  
Cellular Level ◽  
Src Family Kinases ◽  
Temperature Sensitive ◽  
Rate Limiting Step ◽  
Truncation Mutant ◽  
Wild Type Counterpart

ABSTRACT Genetic studies have previously revealed that Cdc37p is required for the catalytic competence of v-Src in yeast. We have reasoned that temperature-sensitive mutants of Src family kinases might be more sensitive to the cellular level of p50Cdc37, the mammalian homolog of Cdc37p, than their wild-type counterpart, thus potentially providing a unique opportunity to elucidate the involvement of p50Cdc37 in the folding and stabilization of Src family kinases. A temperature-sensitive mutant of a constitutively active form of Hck (i.e., tsHck499F) was created by mutating two amino acids within the kinase domain of Hck499F. Significantly, overexpression of p50Cdc37 rescues the catalytic activity of tsHck499F at 33°C, while partially buffering it against inactivation at higher temperatures (e.g., 37 and 39°C). Hsp90 function is required for tsHck499F activity and its stabilization by p50Cdc37, but overexpression of Hsp90 is not sufficient to stabilize tsHck499F. Overexpression of p50Cdc37 promotes the association of tsHck499F with Hsp90, suggesting that the cellular level of p50Cdc37might be the rate-limiting step in the association oftsHck499F with Hsp90. A truncation mutant of p50Cdc37 that cannot bind Hsp90 still has a limited capacity to rescue the catalytic activity of tsHck499F and promote its association with Hsp90. This is a particularly important observation, since it argues that rather than solely acting as a passive adapter protein to tether tsHck499F to Hsp90, p50Cdc37 may also act allosterically to enhance the association of tsHck499F with Hsp90. The findings presented here might also have implications for our understanding of the evolution of protein kinases and tumor development.

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