scholarly journals Inositol acylation of glycosylphosphatidylinositols in the pathogenic fungus Cryptococcus neoformansz and the model yeast Saccharomyces cerevisiae

1999 ◽  
Vol 340 (1) ◽  
pp. 25-32 ◽  
Author(s):  
Sarah P. FRANZOT ◽  
Tamara L. DOERING
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Mammalian Cells ◽  
Pathogenic Fungus ◽  
Sugar Residue ◽  
Yeast Saccharomyces Cerevisiae ◽  
Free System ◽  
Life Threatening

Cryptococcus neoformans, an opportunistic fungus responsible for life-threatening infection in immunocompromised patients, is able to synthesize glycosylphosphatidylinositol (GPI) structures. Radiolabelling experiments in vitro with the use of a cryptococcal cell-free system showed that the pathway begins as in other eukaryotes, with the addition of N-acetylglucosamine to phosphatidylinositol, followed by deacetylation of the sugar residue. The third step, acylation of the inositol ring, seemed to involve a fatty acid other than palmitate, in contrast with previous findings in Saccharomyces cerevisiae and mammalian GPI pathways. A systematic study of inositol acylation in C. neoformans and S. cerevisiae showed that both organisms used a variety of fatty acids in this step; these were transferred directly from acyl-CoA to inositol without modification. However, the specificity of fatty acid utilization was quite distinct in the two fungi, with the pathogen being substantially more restrictive. In mammalian cells fatty acids added exogenously as acyl-CoAs are not transferred directly to inositol. These results suggest significant differences in the GPI biosynthetic pathway between mammalian and C. neoformans cells that could represent targets for anti-cryptococcal therapy.

scholarly journals Characterization of sphingosine kinase (SK) activity in Saccharomyces cerevisiae and isolation of SK-deficient mutants

1998 ◽  
Vol 332 (2) ◽  
pp. 525-531 ◽  
Author(s):  
Margaret M. LANTERMAN ◽  
Julie D. SABA
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Growth Regulation ◽  
Sphingosine Kinase ◽  
Heat Sensitivity ◽  
Sphingoid Bases ◽  
Yeast Saccharomyces Cerevisiae ◽  
Abnormal Growth

Sphingosine kinase (SK) catalyses the phosphorylation of sphingosine to generate sphingosine 1-phosphate, which is a second messenger involved in the proliferative responses of mammalian cells. Although the yeast Saccharomyces cerevisiae has similar phosphorylated sphingoid bases which appear to be involved in growth regulation and the response to stress, SK activity had not been previously demonstrated in yeast. In this study, an in vitro system was set up to characterize yeast SK activity. Activity was detected in the cytosol at neutral pH and 37 °C. Yeast SK phosphorylated the sphingoid bases sphingosine, dihydrosphingosine and phytosphingosine. (d,l)-threo-dihydrosphingosine, an inhibitor of mammalian SK, did not inhibit the yeast enzyme. Unique properties of yeast SK were an optimal temperature of 43 °C, and in vivo activation during nutrient deprivation. Spontaneous mutants with diminished SK activity were isolated utilizing a screen for resistance to sphingosine in a sphingosine-phosphate-lyase deletion background. Abnormal growth and heat sensitivity were observed in these mutants. These findings suggest that SK may function as a stress-response protein in yeast.

scholarly journals Hypothesis: cell volume limits cell divisions.

2006 ◽  
Vol 53 (4) ◽  
pp. 833-835 ◽  
Author(s):  
Tomasz Biliński ◽  
Grzegorz Bartosz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Volume ◽  
Mammalian Cells ◽  
Somatic Cells ◽  
Large Cell ◽  
Yeast Cells ◽  
Replicative Aging ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cell Divisions

Mammalian somatic cells and also cells of the yeast Saccharomyces cerevisiae are capable of undergoing a limited number of divisions. Reaching the division limit is referred to, apparently not very fortunately, as replicative aging. A common feature of S. cerevisiae cells and fibroblasts approaching the limit of cell divisions in vitro is attaining giant volumes. In yeast cells this phenomenon is an inevitable consequence of budding so it is not causally related to aging. Therefore, reaching a critically large cell volume may underlie the limit of cell divisions. A similar phenomenon may limit the number of cell divisions of cultured mammalian cells. The term replicative (generative) aging may be therefore illegitimate.

Expression and characterization of ras mRNAs from Saccharomyces cerevisiae

1984 ◽  
Vol 4 (11) ◽  
pp. 2298-2305
Author(s):  
G L Temeles ◽  
D DeFeo-Jones ◽  
K Tatchell ◽  
M S Ellinger ◽  
E M Scolnick
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Cross Reactivity ◽  
Binding Activity ◽  
In Vitro Translation ◽  
Ras Gene ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ras Genes ◽  
In The Wild

The cellular homologs of the Harvey and Kirsten murine sarcoma virus oncogenes comprise a multigene family, ras, that displays striking evolutionary conservation. We recently reported [DeFeo-Jones et al., Nature (London) 306:707-709, 1983] the cloning of two ras homologs from the yeast Saccharomyces cerevisiae. The nucleotide sequences of these genes predict polypeptides that show remarkable homology to p21, the mammalian ras gene product. We have also found proteins in yeast lysates with serological cross-reactivity to p21 (Papageorge et al., Mol. Cell. Biol. 4:23-29, 1984). In this work, we explored the relationship between the immunoprecipitated proteins and the yeast ras genes. We show that both ras genes are expressed in the wild-type cell. Furthermore, we demonstrate by in vitro translation of hybrid-selected RASsc1 mRNA and immunoprecipitation of the translation products that the cloned RASsc1 gene encodes the proteins immunoprecipitated from yeast lysates by anti-p21 monoclonal antibody. Finally, we used anti-p21 monoclonal antibodies to detect a guanine nucleotide binding activity in yeast lysates. The structural and biochemical homologies between ras gene products of S. cerevisiae and mammalian cells suggest that information obtained by genetic analysis of ras function in a lower eucaryote should be applicable to higher organisms as well.

scholarly journals Expression and characterization of ras mRNAs from Saccharomyces cerevisiae.

1984 ◽  
Vol 4 (11) ◽  
pp. 2298-2305 ◽  
Author(s):  
G L Temeles ◽  
D DeFeo-Jones ◽  
K Tatchell ◽  
M S Ellinger ◽  
E M Scolnick
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Cross Reactivity ◽  
Binding Activity ◽  
In Vitro Translation ◽  
Ras Gene ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ras Genes ◽  
In The Wild

The cellular homologs of the Harvey and Kirsten murine sarcoma virus oncogenes comprise a multigene family, ras, that displays striking evolutionary conservation. We recently reported [DeFeo-Jones et al., Nature (London) 306:707-709, 1983] the cloning of two ras homologs from the yeast Saccharomyces cerevisiae. The nucleotide sequences of these genes predict polypeptides that show remarkable homology to p21, the mammalian ras gene product. We have also found proteins in yeast lysates with serological cross-reactivity to p21 (Papageorge et al., Mol. Cell. Biol. 4:23-29, 1984). In this work, we explored the relationship between the immunoprecipitated proteins and the yeast ras genes. We show that both ras genes are expressed in the wild-type cell. Furthermore, we demonstrate by in vitro translation of hybrid-selected RASsc1 mRNA and immunoprecipitation of the translation products that the cloned RASsc1 gene encodes the proteins immunoprecipitated from yeast lysates by anti-p21 monoclonal antibody. Finally, we used anti-p21 monoclonal antibodies to detect a guanine nucleotide binding activity in yeast lysates. The structural and biochemical homologies between ras gene products of S. cerevisiae and mammalian cells suggest that information obtained by genetic analysis of ras function in a lower eucaryote should be applicable to higher organisms as well.

scholarly journals The RAD7, RAD16, and RAD23 genes of Saccharomyces cerevisiae: requirement for transcription-independent nucleotide excision repair in vitro and interactions between the gene products.

1997 ◽  
Vol 17 (2) ◽  
pp. 635-643 ◽  
Author(s):  
Z Wang ◽  
S Wei ◽  
S H Reed ◽  
X Wu ◽  
J Q Svejstrup ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleotide Excision Repair ◽  
Protein Complex ◽  
Excision Repair ◽  
Dna Lesions ◽  
Yeast Saccharomyces Cerevisiae ◽  
Free System ◽  
Nucleotide Excision ◽  
A Cell

Nucleotide excision repair (NER) is a biochemical process required for the repair of many different types of DNA lesions. In the yeast Saccharomyces cerevisiae, the RAD7, RAD16, and RAD23 genes have been specifically implicated in NER of certain transcriptionally repressed loci and in the nontranscribed strand of transcriptionally active genes. We have used a cell-free system to study the roles of the Rad7, Rad16, and Rad23 proteins in NER. Transcription-independent NER of a plasmid substrate was defective in rad7, rad16, and rad23 mutant extracts. Complementation studies with a previously purified NER protein complex (nucleotide excision repairosome) indicate that Rad23 is a component of the repairosome, whereas Rad7 and Rad16 proteins were not found in this complex. Complementation studies with rad4, rad7, rad16, and rad23 mutant extracts suggest physical interactions among these proteins. This conclusion was confirmed by experiments using the yeast two-hybrid assay, which demonstrated the following pairwise interactions: Rad4 with Rad23, Rad4 with Rad7, and Rad7 with Rad16. Additionally, interaction between the Rad7 and Rad16 proteins was demonstrated in vitro. Our results show that Rad7, Rad16, and Rad23 are required for transcription-independent NER in vitro. This process may involve a unique protein complex which is distinct from the repairosome and which contains at least the Rad4, Rad7, and Rad16 proteins.

scholarly journals mBet3p is required for homotypic COPII vesicle tethering in mammalian cells

2006 ◽  
Vol 174 (3) ◽  
pp. 359-368 ◽  
Author(s):  
Sidney Yu ◽  
Ayano Satoh ◽  
Marc Pypaert ◽  
Karl Mullen ◽  
Jesse C. Hay ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Vesicle Tethering ◽  
Transitional Endoplasmic Reticulum ◽  
Large Complex ◽  
Copii Vesicle ◽  
Copii Vesicles

TRAPPI is a large complex that mediates the tethering of COPII vesicles to the Golgi (heterotypic tethering) in the yeast Saccharomyces cerevisiae. In mammalian cells, COPII vesicles derived from the transitional endoplasmic reticulum (tER) do not tether directly to the Golgi, instead, they appear to tether to each other (homotypic tethering) to form vesicular tubular clusters (VTCs). We show that mammalian Bet3p (mBet3p), which is the most highly conserved TRAPP subunit, resides on the tER and adjacent VTCs. The inactivation of mBet3p results in the accumulation of cargo in membranes that colocalize with the COPII coat. Furthermore, using an assay that reconstitutes VTC biogenesis in vitro, we demonstrate that mBet3p is required for the tethering and fusion of COPII vesicles to each other. Consistent with the proposal that mBet3p is required for VTC biogenesis, we find that ERGIC-53 (VTC marker) and Golgi architecture are disrupted in siRNA-treated mBet3p-depleted cells. These findings imply that the TRAPPI complex is essential for VTC biogenesis.

scholarly journals Antifungal activity of dendritic cell lysosomal proteins against Cryptococcus neoformans

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Benjamin N. Nelson ◽  
Savannah G. Beakley ◽  
Sierra Posey ◽  
Brittney Conn ◽  
Emma Maritz ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Cryptococcus Neoformans ◽  
Mammalian Cells ◽  
Cysteine Proteases ◽  
Dependent Manner ◽  
Life Threatening ◽  
Opportunistic Fungal Pathogen ◽  
Dose Dependent ◽  
Lysosomal Proteins

AbstractCryptococcal meningitis is a life-threatening disease among immune compromised individuals that is caused by the opportunistic fungal pathogen Cryptococcus neoformans. Previous studies have shown that the fungus is phagocytosed by dendritic cells (DCs) and trafficked to the lysosome where it is killed by both oxidative and non-oxidative mechanisms. While certain molecules from the lysosome are known to kill or inhibit the growth of C. neoformans, the lysosome is an organelle containing many different proteins and enzymes that are designed to degrade phagocytosed material. We hypothesized that multiple lysosomal components, including cysteine proteases and antimicrobial peptides, could inhibit the growth of C. neoformans. Our study identified the contents of the DC lysosome and examined the anti-cryptococcal properties of different proteins found within the lysosome. Results showed several DC lysosomal proteins affected the growth of C. neoformans in vitro. The proteins that killed or inhibited the fungus did so in a dose-dependent manner. Furthermore, the concentration of protein needed for cryptococcal inhibition was found to be non-cytotoxic to mammalian cells. These data show that many DC lysosomal proteins have antifungal activity and have potential as immune-based therapeutics.

scholarly journals Cells Expressing Murine RAD52 Splice Variants Favor Sister Chromatid Repair

2006 ◽  
Vol 26 (10) ◽  
pp. 3752-3763 ◽  
Author(s):  
Peter H. Thorpe ◽  
Vanessa A. Marrero ◽  
Margaret H. Savitzky ◽  
Ivana Sunjevaric ◽  
Tom C. Freeman ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Sister Chromatid ◽  
Mammalian Cells ◽  
Splice Variants ◽  
Protein A ◽  
Single Stranded Dna ◽  
Culture Cells ◽  
Dominant Phenotype ◽  
Mouse Tissues

ABSTRACT The RAD52 gene is essential for homologous recombination in the yeast Saccharomyces cerevisiae. RAD52 is the archetype in an epistasis group of genes essential for DNA damage repair. By catalyzing the replacement of replication protein A with Rad51 on single-stranded DNA, Rad52 likely promotes strand invasion of a double-stranded DNA molecule by single-stranded DNA. Although the sequence and in vitro functions of mammalian RAD52 are conserved with those of yeast, one difference is the presence of introns and consequent splicing of the mammalian RAD52 pre-mRNA. We identified two novel splice variants from the RAD52 gene that are expressed in adult mouse tissues. Expression of these splice variants in tissue culture cells elevates the frequency of recombination that uses a sister chromatid template. To characterize this dominant phenotype further, the RAD52 gene from the yeast Saccharomyces cerevisiae was truncated to model the mammalian splice variants. The same dominant sister chromatid recombination phenotype seen in mammalian cells was also observed in yeast. Furthermore, repair from a homologous chromatid is reduced in yeast, implying that the choice of alternative repair pathways may be controlled by these variants. In addition, a dominant DNA repair defect induced by one of the variants in yeast is suppressed by overexpression of RAD51, suggesting that the Rad51-Rad52 interaction is impaired.

TetR-family transcriptional repressor Thermus thermophilus FadR controls fatty acid degradation

Microbiology ◽  
2011 ◽  
Vol 157 (6) ◽  
pp. 1589-1601 ◽  
Author(s):  
Yoshihiro Agari ◽  
Kazuko Agari ◽  
Keiko Sakamoto ◽  
Seiki Kuramitsu ◽  
Akeo Shinkai
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Target Genes ◽  
Thermus Thermophilus ◽  
Acyl Chain ◽  
Metabolic Energy ◽  
Straight Chain ◽  
Fatty Acid Degradation ◽  
Acid Degradation

In the extremely thermophilic bacterium Thermus thermophilus HB8, one of the four TetR-family transcriptional regulators, which we named T. thermophilus FadR, negatively regulated the expression of several genes, including those involved in fatty acid degradation, both in vivo and in vitro. T. thermophilus FadR repressed the expression of the target genes by binding pseudopalindromic sequences covering the predicted −10 hexamers of their promoters, and medium-to-long straight-chain (C10–18) fatty acyl-CoA molecules were effective for transcriptional derepression. An X-ray crystal structure analysis revealed that T. thermophilus FadR bound one lauroyl (C12)-CoA molecule per FadR monomer, with its acyl chain moiety in the centre of the FadR molecule, enclosed within a tunnel-like substrate-binding pocket surrounded by hydrophobic residues, and the CoA moiety interacting with basic residues on the protein surface. The growth of T. thermophilus HB8, with palmitic acid as the sole carbon source, increased the expression of FadR-regulated genes. These results indicate that in T. thermophilus HB8, medium-to-long straight-chain fatty acids can be used for metabolic energy under the control of FadR, although the major fatty acids found in this strain are iso- and anteiso-branched-chain (C15 and 17) fatty acids.

scholarly journals The Highly Conserved tRNAHis Guanylyltransferase Thg1p Interacts with the Origin Recognition Complex and Is Required for the G2/M Phase Transition in the Yeast Saccharomyces cerevisiae

2005 ◽  
Vol 4 (4) ◽  
pp. 832-835 ◽  
Author(s):  
Terri S. Rice ◽  
Min Ding ◽  
David S. Pederson ◽  
Nicholas H. Heintz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Origin Recognition Complex ◽  
Nuclear Division ◽  
Permissive Temperature ◽  
Yeast Saccharomyces Cerevisiae ◽  
M Phase ◽  
And Migration ◽  
Origin Recognition

ABSTRACT Here we show that the Saccharomyces cerevisiae tRNAHis guanylyltransferase Thg1p interacts with the origin recognition complex in vivo and in vitro and that overexpression of hemagglutinin-Thg1p selectively impedes growth of orc2-1(Ts) cells at the permissive temperature. Studies with conditional mutants indicate that Thg1p couples nuclear division and migration to cell budding and cytokinesis in yeast.

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