Intracellular Signal Triggered by Cholera Toxin inSaccharomyces boulardii and Saccharomyces cerevisiae

1998 ◽  
Vol 64 (2) ◽  
pp. 564-568 ◽  
Author(s):  
Rogelio L. Brandão ◽  
Ieso M. Castro ◽  
Eduardo A. Bambirra ◽  
Sheila C. Amaral ◽  
Luciano G. Fietto ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cholera Toxin ◽  
Mammalian Cells ◽  
Specific Binding ◽  
Saccharomyces Boulardii ◽  
Yeast Cells ◽  
B Subunit ◽  
Nonfermentable Carbon Source ◽  
A Subunit ◽  
Yeast Surface

ABSTRACT As is the case for Saccharomyces boulardii, Saccharomyces cerevisiae W303 protects Fisher rats against cholera toxin (CT). The addition of glucose or dinitrophenol to cells of S. boulardii grown on a nonfermentable carbon source activated trehalase in a manner similar to that observed for S. cerevisiae. The addition of CT to the same cells also resulted in trehalase activation. Experiments performed separately on the A and B subunits of CT showed that both are necessary for activation. Similarly, the addition of CT but not of its separate subunits led to a cyclic AMP (cAMP) signal in both S. boulardii and S. cerevisiae. These data suggest that trehalase stimulation by CT probably occurred through the cAMP-mediated protein phosphorylation cascade. The requirement of CT subunit B for both the cAMP signal and trehalase activation indicates the presence of a specific receptor on the yeasts able to bind to the toxin, a situation similar to that observed for mammalian cells. This hypothesis was reinforced by experiments with 125I-labeled CT showing specific binding of the toxin to yeast cells. The adhesion of CT to a receptor on the yeast surface through the B subunit and internalization of the A subunit (necessary for the cAMP signal and trehalase activation) could be one more mechanism explaining protection against the toxin observed for rats treated with yeasts.

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (9) ◽  
pp. 4084-4092
Author(s):  
P C McCabe ◽  
H Haubruck ◽  
P Polakis ◽  
F McCormick ◽  
M A Innis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Bound States ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Amino Terminal ◽  
Loop Region

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

scholarly journals Differential Biological and Adjuvant Activities of Cholera Toxin and Escherichia coli Heat-Labile Enterotoxin Hybrids

2001 ◽  
Vol 69 (3) ◽  
pp. 1528-1535 ◽  
Author(s):  
Christal C. Bowman ◽  
John D. Clements
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Cholera Toxin ◽  
The Other ◽  
Toxin Gene ◽  
Wild Type ◽  
B Subunit ◽  
Heat Labile ◽  
A Subunit ◽  
Ifn Γ

ABSTRACT Two bacterial products that have been demonstrated to function as mucosal adjuvants are cholera toxin (CT), produced by various strains of Vibrio cholerae, and the heat-labile enterotoxin (LT) produced by some enterotoxigenic strains of Escherichia coli. Although LT and CT have many features in common, they are clearly distinct molecules with biochemical and immunologic differences which make them unique. The goal of this study was to determine the basis for these biological differences by constructing and characterizing chimeric CT-LT molecules. Toxin gene fragments were subcloned to create two constructs, each expressing the enzymatically active A subunit of one toxin and the receptor binding B subunit of the other toxin. These hybrid toxins were purified, and the composition and assembly of CT A subunit (CT-A)-LT B subunit (LT-B) and LT A subunit (LT-A)-CT B subunit (CT-B) were confirmed. Hybrids were evaluated for enzymatic activity, as measured by the accumulation of cyclic AMP in Caco-2 cells, and the enterotoxicity of each toxin was assessed in a patent-mouse assay. The results demonstrated that LT-A–CT-B induces the accumulation of lower levels of cyclic AMP and has less enterotoxicity than either wild-type toxin or the other hybrid. Nonetheless, this hybrid retains adjuvant activity equivalent to or greater than that of either wild-type toxin or the other hybrid when used in conjunction with tetanus toxoid for intranasal immunization of BALB/c mice. Importantly, the ability of LT to induce a type 1 cytokine response was found to be a function of LT-A. Specifically, LT-A–CT-B was able to augment the levels of antigen-specific gamma interferon (IFN-γ) and interleukin 5 to levels comparable to those achieved with native LT, while CT-A–LT-B and native CT both produced lower levels of antigen-specific IFN-γ. Thus, these toxin hybrids possess unique biological characteristics and provide information about the basis for differences in the biological activities observed for CT and LT.

scholarly journals RSR1, a ras-like gene homologous to Krev-1 (smg21A/rap1A): role in the development of cell polarity and interactions with the Ras pathway in Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (2) ◽  
pp. 758-766 ◽  
Author(s):  
R Ruggieri ◽  
A Bender ◽  
Y Matsui ◽  
S Powers ◽  
Y Takai ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Polarity ◽  
Copy Number ◽  
Mammalian Cells ◽  
Direct Interaction ◽  
Dominant Negative ◽  
Yeast Cells ◽  
Wild Type ◽  
Ras Gene ◽  
Ras Pathway

The Saccharomyces cerevisiae ras-like gene RSR1 is particularly closely related to the mammalian gene Krev-1 (also known as smg21A and rap1A). RSR1 was originally isolated as a multicopy suppressor of a cdc24 mutation, which causes an inability to bud or establish cell polarity. Deletion of RSR1 itself does not affect growth but causes a randomization of bud position. We have now constructed mutant alleles of RSR1 encoding proteins with substitutions of Val for Gly at position 12 (analogous to constitutively activated Ras proteins) or Asn for Lys at position 16 (analogous to a dominant-negative Ras protein). rsr1Val-12 could not restore a normal budding pattern to an rsr1 deletion strain but could suppress a cdc24 mutation when overexpressed. rsr1Asn-16 could randomize the budding pattern of a wild-type strain even in low copy number but was not lethal even in high copy number. These and other results suggest that Rsr1p functions only in bud site selection and not in subsequent events of polarity establishment and bud formation, that this function involves a cycling between GTP-bound and GDP-bound forms of the protein, and that the suppression of cdc24 involves direct interaction between Rsr1p[GTP] and Cdc24p. Functional homology between Rsr1p and Krev-1 p21 was suggested by the observations that expression of the latter protein in yeast cells could both suppress a cdc24 mutation and randomize the budding pattern of wild-type cells. As Krev-1 overexpression can suppress ras-induced transformation of mammalian cells, we looked for effects of RSR1 on the S. cerevisiae Ras pathway. Although no suppression of the activated RAS2Val-19 allele was observed, overexpression of rsr1Val-12 suppressed the lethality of strains lacking RAS gene function, apparently through a direct activation of adenyl cyclase. This interaction of Rsr1p with the effector of Ras in S. cerevisiae suggests that Krev-1 may revert ras-induced transformation of mammalian cells by affecting the interaction of ras p21 with its effector.

scholarly journals Nuclear RNase MRP is required for correct processing of pre-5.8S rRNA in Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (12) ◽  
pp. 7935-7941 ◽  
Author(s):  
M E Schmitt ◽  
D A Clayton
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Yeast Cells ◽  
Rrna Processing ◽  
Rnase Mrp ◽  
Gal1 Promoter ◽  
5.8S Rrna ◽  
Late Step ◽  
Conditional Mutations ◽  
A Site

RNase MRP is a site-specific ribonucleoprotein endoribonuclease that cleaves RNA from the mitochondrial origin of replication in a manner consistent with a role in priming leading-strand DNA synthesis. Despite the fact that the only known RNA substrate for this enzyme is complementary to mitochondrial DNA, the majority of the RNase MRP activity in a cell is found in the nucleus. The recent characterization of this activity in Saccharomyces cerevisiae and subsequent cloning of the gene coding for the RNA subunit of the yeast enzyme have enabled a genetic approach to the identification of a nuclear role for this ribonuclease. Since the gene for the RNA component of RNase MRP, NME1, is essential in yeast cells and RNase MRP in mammalian cells appears to be localized to nucleoli within the nucleus, we utilized both regulated expression and temperature-conditional mutations of NME1 to assay for a possible effect on rRNA processing. Depletion of the RNA component of the enzyme was accomplished by using the glucose-repressed GAL1 promoter. Shortly after the shift to glucose, the RNA component of the enzyme was found to be depleted severely, and rRNA processing was found to be normal at all sites except the B1 processing site. The B1 site, at the 5' end of the mature 5.8S rRNA, is actually composed of two cleavage sites 7 nucleotides apart. This cleavage normally generates two species of 5.8S rRNA at a ratio of 10:1 (small to large) in most eukaryotes. After RNase MRP depletion, yeast cells were found to have almost exclusively the larger species of 5.8S rRNA. In addition, an aberrant 309-nucleotide precursor that stretched from the A2 to E processing sites of rRNA accumulated in these cells. Temperature-conditional mutations in the RNase MRP RNA gene gave an identical phenotype.Translation in yeast cells depleted of the smaller 5.8S rRNA was found to remain robust, suggesting a possible function for two 5.8S rRNAs in the regulated translation of select messages. These results are consistent with RNase MRP playing a role in a late step of rRNA processing. The data also indicate a requirement for having the smaller form of 5.8S rRNA, and they argue for processing at the B1 position being composed of two separate cleavage events catalyzed by two different activities.

scholarly journals Gangliosides That Associate with Lipid Rafts Mediate Transport of Cholera and Related Toxins from the Plasma Membrane to Endoplasmic Reticulm

2003 ◽  
Vol 14 (12) ◽  
pp. 4783-4793 ◽  
Author(s):  
Yukako Fujinaga ◽  
Anne A. Wolf ◽  
Chiara Rodighiero ◽  
Heidi Wheeler ◽  
Billy Tsai ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cholera Toxin ◽  
Lipid Rafts ◽  
Intestinal Cells ◽  
B Subunit ◽  
High Affinity ◽  
A Subunit ◽  
Human Intestinal Cells

Cholera toxin (CT) travels from the plasma membrane of intestinal cells to the endoplasmic reticulum (ER) where a portion of the A-subunit, the A1 chain, crosses the membrane into the cytosol to cause disease. A related toxin, LTIIb, binds to intestinal cells but does not cause toxicity. Here, we show that the B-subunit of CT serves as a carrier for the A-subunit to the ER where disassembly occurs. The B-subunit binds to gangliosides in lipid rafts and travels with the ganglioside to the ER. In many cells, LTIIb follows a similar pathway, but in human intestinal cells it binds to a ganglioside that fails to associate with lipid rafts and it is sorted away from the retrograde pathway to the ER. Our results explain why LTIIb does not cause disease in humans and suggest that gangliosides with high affinity for lipid rafts may provide a general vehicle for the transport of toxins to the ER.

Anthrax toxin-mediated delivery of cholera toxin-A subunit into the cytosol of mammalian cells

2000 ◽  
Vol 32 (1) ◽  
pp. 69 ◽  
Author(s):  
Manju Sharma ◽  
Hemant Khanna ◽  
Naveen Arora ◽  
Yogendra Singh
Keyword(s):  
Cholera Toxin ◽  
Mammalian Cells ◽  
Anthrax Toxin ◽  
Toxin A ◽  
A Subunit

Adenovirus transcriptional regulatory regions are conserved in mammalian cells and Saccharomyces cerevisiae

1988 ◽  
Vol 8 (9) ◽  
pp. 3717-3725
Author(s):  
M Kornuc ◽  
R Altman ◽  
D Harrich ◽  
J Garcia ◽  
J Chao ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Transcriptional Regulation ◽  
Binding Sites ◽  
Mammalian Cells ◽  
Yeast Cells ◽  
Binding Domains ◽  
Cell Extracts ◽  
Dnase I Footprinting ◽  
Transactivator Protein

The adenovirus early region 3 (E3) promoter is an early viral promoter which is strongly induced by the adenovirus transactivator protein E1A. DNase I footprinting with HeLa cell extracts has identified four factor-binding domains which appear to be involved in basal and E1A-induced transcriptional regulation. These binding domains may bind TATA region-binding factors (site I), the CREB/ATF protein (site II), the AP-1 protein (site III), and nuclear factor I/CTF (site IV). Recently, it has been shown that the DNA-binding domain of transcription factor AP-1 has homology with the yeast transcription factor GCN4 and that the yeast transactivator protein GAL4 is able to stimulate transcription in HeLa cells from promoters containing GAL4-binding sites. These results suggest an evolutionary conservation of both transcription factors and the mechanisms responsible for transcriptional activation in Saccharomyces cerevisiae and higher eucaryotic organisms. To determine whether similar patterns of transcriptional regulation were seen with the E3 promoter in HeLa and yeast cells, the E3 promoter fused to the chloramphenicol acetyltransferase (cat) gene was cloned into a high-copy-number plasmid and stably introduced into yeast cells. S1 analysis revealed that similar E3 promoter mRNA start sites were found in yeast and HeLa cells. DNase I footprinting with partially purified yeast extracts revealed that four regions of the E3 promoter were protected. Several of these regions were similar to binding sites determined by using HeLa cell extracts. Oligonucleotide mutagenesis of these binding domains indicated their importance in the transcriptional regulation of the E3 promoter in yeast cells. These results suggest that similar cellular transcription factor-binding sites may be involved in the regulation of promoters in both yeast and mammalian cells.

Citrate synthase encoded by the CIT2 gene of Saccharomyces cerevisiae is peroxisomal

1990 ◽  
Vol 10 (4) ◽  
pp. 1399-1405
Author(s):  
A S Lewin ◽  
V Hines ◽  
G M Small
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Citrate Synthase ◽  
Glyoxylate Cycle ◽  
Mitochondrial Fraction ◽  
Yeast Cells ◽  
Mitochondrial Activity ◽  
Major Peak ◽  
Gene Encoding ◽  
Peroxisomal Targeting

The product of the CIT2 gene has the tripeptide SKL at its carboxyl terminus. This amino acid sequence has been shown to act as a peroxisomal targeting signal in mammalian cells. We examined the subcellular site of this extramitochondrial citrate synthase. Cells of Saccharomyces cerevisiae were grown on oleate medium to induce peroxisome proliferation. A fraction containing membrane-enclosed vesicles and organelles was analyzed by sedimentation on density gradients. In wild-type cells, the major peak of citrate synthase activity was recovered in the mitochondrial fraction, but a second peak of activity cosedimented with peroxisomes. The peroxisomal activity, but not the mitochondrial activity, was inhibited by incubation at pH 8.1, a characteristic of the extramitochondrial citrate synthase encoded by the CIT2 gene. In a strain in which the CIT1 gene encoding mitochondrial citrate synthase had been disrupted, the major peak of citrate synthase activity was peroxisomal, and all of the activity was sensitive to incubation at pH 8.1. Yeast cells bearing a cit2 disruption were unable to mobilize stored lipids and did not form stable peroxisomes in oleate. We conclude that citrate synthase encoded by CIT2 is peroxisomal and participates in the glyoxylate cycle.

scholarly journals Signal recognition particle receptor is important for cell growth and protein secretion in Saccharomyces cerevisiae.

1992 ◽  
Vol 3 (8) ◽  
pp. 895-911 ◽  
Author(s):  
S C Ogg ◽  
M A Poritz ◽  
P Walter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Growth ◽  
Mammalian Cells ◽  
Protein Targeting ◽  
Signal Recognition Particle ◽  
Yeast Cells ◽  
Secretory Proteins ◽  
Signal Recognition ◽  
Er Membrane

In mammalian cells, the signal recognition particle (SRP) receptor is required for the targeting of nascent secretory proteins to the endoplasmic reticulum (ER) membrane. We have identified the Saccharomyces cerevisiae homologue of the alpha-subunit of the SRP receptor (SR alpha) and characterized its function in vivo. S. cerevisiae SR alpha is a 69-kDa peripheral membrane protein that is 32% identical (54% chemically similar) to its mammalian homologue and, like mammalian SR alpha, is predicted to contain a GTP binding domain. Yeast cells that contain the SR alpha gene (SRP101) under control of the GAL1 promoter show impaired translocation of soluble and membrane proteins across the ER membrane after depletion of SR alpha. The degree of the translocation defect varies for different proteins. The defects are similar to those observed in SRP deficient cells. Disruption of the SRP101 gene results in an approximately sixfold reduction in the growth rate of the cells. Disruption of the gene encoding SRP RNA (SCR1) or both SCR1 and SRP101 resulted in an indistinguishable growth phenotype, indicating that SRP receptor and SRP function in the same pathway. Taken together, these results suggest that the components and the mechanism of the SRP-dependent protein targeting pathway are evolutionarily conserved yet not essential for cell growth. Surprisingly, cells that are grown for a prolonged time in the absence of SRP or SRP receptor no longer show pronounced protein translocation defects. This adaptation is a physiological process and is not due to the accumulation of a suppressor mutation. The degree of this adaptation is strain dependent.

scholarly journals Nodamura Virus RNA Replication in Saccharomyces cerevisiae: Heterologous Gene Expression Allows Replication-Dependent Colony Formation

2005 ◽  
Vol 79 (1) ◽  
pp. 495-502 ◽  
Author(s):  
B. Duane Price ◽  
Lance D. Eckerle ◽  
L. Andrew Ball ◽  
Kyle L. Johnson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Heterologous Gene Expression ◽  
Rna Replication ◽  
Viral Rna ◽  
Yeast Cells ◽  
Genomic Rnas ◽  
Nodamura Virus

ABSTRACT Nodamura virus (NoV) and Flock House virus (FHV) are members of the family Nodaviridae. The nodavirus genome is composed of two positive-sense RNA segments: RNA1 encodes the viral RNA-dependent RNA polymerase and RNA2 encodes the capsid protein precursor. A small subgenomic RNA3, which encodes nonstructural proteins B1 and B2, is transcribed from RNA1 during RNA replication. Previously, FHV was shown to replicate both of its genomic RNAs and to transcribe RNA3 in transiently transfected yeast cells. FHV RNAs and their derivatives could also be expressed from plasmids containing RNA polymerase II promoters. Here we show that all of these features can be recapitulated for NoV, the only nodavirus that productively infects mammals. Inducible plasmid-based systems were used to characterize the RNA replication requirements for NoV RNA1 and RNA2 in Saccharomyces cerevisiae. Induced NoV RNA1 replication was robust. Three previously described NoV RNA1 mutants behaved in yeast as they had in mammalian cells. Yeast colonies were selected from cells expressing NoV RNA1, and RNA2 replicons that encoded yeast nutritional markers, from plasmids. Unexpectedly, these NoV RNA replication-dependent yeast colonies were recovered at frequencies 104-fold lower than in the analogous FHV system. Molecular analysis revealed that some of the NoV RNA replication-dependent colonies contained mutations in the NoV B2 open reading frame in the replicating viral RNA. In addition, we found that NoV RNA1 could support limited replication of a deletion derivative of the heterologous FHV RNA2 that expressed the yeast HIS3 selectable marker, resulting in formation of HIS+ colonies.

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