scholarly journals Cytological analysis of Saccharomyces cerevisiae cells supporting cymbidium ringspot virus defective interfering RNA replication

2006 ◽  
Vol 87 (3) ◽  
pp. 705-714 ◽  
Author(s):  
Beatriz Navarro ◽  
Marcello Russo ◽  
Vitantonio Pantaleo ◽  
Luisa Rubino
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Infected Plant ◽  
Rna Replication ◽  
Ringspot Virus ◽  
Yeast Cells ◽  
Replicase Proteins ◽  
Defective Interfering Rna ◽  
Interfering Rna

The replicase proteins p33 and p92 of Cymbidium ringspot virus (CymRSV) were found to support the replication of defective interfering (DI) RNA in Saccharomyces cerevisiae cells. Two yeast strains were used, differing in the biogenesis of peroxisomes, the organelles supplying the membranous vesicular environment in which CymRSV RNA replication takes place in infected plant cells. Double-labelled immunofluorescence showed that both p33 and p92 replicase proteins localized to peroxisomes, independently of one another and of the presence of the replication template. It is suggested that these proteins are sorted initially from the cytosol to the endoplasmic reticulum and then to peroxisomes. However, only the expression of p33, but not p92, increased the number of peroxisomes and induced membrane proliferation. DI RNA replication occurred in yeast cells, as demonstrated by the presence of monomers and dimers of positive and negative polarities. Labelling with BrUTP showed that peroxisomes were the sites of nascent viral synthesis, whereas in situ hybridization indicated that DI RNA progeny were diffused throughout the cytoplasm. DI RNA replication also took place in yeast cells devoid of peroxisomes. It is suggested that replication in these cells was targeted to the endoplasmic reticulum.

scholarly journals Cymbidium ringspot virus defective interfering RNA replication in yeast cells occurs on endoplasmic reticulum-derived membranes in the absence of peroxisomes

2007 ◽  
Vol 88 (5) ◽  
pp. 1634-1642 ◽  
Author(s):  
Luisa Rubino ◽  
Beatriz Navarro ◽  
Marcello Russo
Keyword(s):  
Endoplasmic Reticulum ◽  
Infected Plant ◽  
Rna Replication ◽  
Ringspot Virus ◽  
Yeast Cells ◽  
Peroxisomal Membrane ◽  
Novel Approach ◽  
Strict Requirement ◽  
Alternative Sites ◽  
Interfering Rna

The replication of Cymbidium ringspot virus (CymRSV) defective interfering (DI) RNA in cells of the yeast Saccharomyces cerevisiae normally takes place in association with the peroxisomal membrane, thus paralleling the replication events in infected plant cells. However, previous results with a peroxisome-deficient mutant strain of yeast had suggested that the presence of peroxisomes is not a strict requirement for CymRSV DI RNA replication. Thus, a novel approach was used to study the putative alternative sites of replication by using S. cerevisiae strain YPH499 which does not contain normal peroxisomes. In this strain, CymRSV p33 and p92 accumulated over portions of the nuclear membrane and on membranous overgrowths which were identified as endoplasmic reticulum (ER) strands, following immunofluorescence and immunoelectron microscope observations. The proteins were not released by high-pH treatment, but were susceptible to proteolytic digestion, thus indicating peripheral and not integrated association. ER-associated p33 and p92 proteins supported in trans the replication of DI RNA. The capacity of plus-strand RNA viruses to replicate in association with different types of cell membranes was thus confirmed.

scholarly journals Replication of Carnation Italian Ringspot Virus Defective Interfering RNA in Saccharomyces cerevisiae

2003 ◽  
Vol 77 (3) ◽  
pp. 2116-2123 ◽  
Author(s):  
Vitantonio Pantaleo ◽  
Luisa Rubino ◽  
Marcello Russo
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ringspot Virus ◽  
Yeast Cells ◽  
Rna Transcripts ◽  
Gal1 Promoter ◽  
Viral Replicase ◽  
Defective Interfering Rna ◽  
Interfering Rna ◽  
Adh1 Promoter

ABSTRACT Two plasmids from which the sequences coding for the 36- and 95-kDa proteins of Carnation Italian ringspot virus (CIRV) could be transcribed in vivo in the yeast Saccharomyces cerevisiae under the control of the ADH1 promoter and terminator were constructed. The two proteins, which constitute the viral replicase, were correctly translated and integrated into membranes of the yeast cells. An additional plasmid was introduced in yeasts expressing the CIRV replicase, from which a defective interfering (DI) RNA (DI-7 RNA) could be transcribed under the control of the GAL1 promoter and terminated by the Tobacco ringspot virus satellite ribozyme, which cleaved 19 nucleotides downstream of the 3′ end of DI RNA. The DI-7 RNA transcripts were amplified by the viral replicase as demonstrated by the restoration of the authentic 3′ end, the requirement of a specific cis-acting signal at this terminus, the preferential accumulation of molecules with the authentic 5′ terminus (AGAAA), the synthesis of head-to-tail dimers, the presence of negative strands, and the incorporation of 5-bromo-UTP. Additionally, transformation with a dimeric construct of DI-7 RNA led to the synthesis of monomers, mimicking the activity of the viral replicase in plant cells.

scholarly journals The p36 and p95 replicase proteins of Carnation Italian ringspot virus cooperate in stabilizing defective interfering RNA

2004 ◽  
Vol 85 (8) ◽  
pp. 2429-2433 ◽  
Author(s):  
Vitantonio Pantaleo ◽  
Luisa Rubino ◽  
Marcello Russo
Keyword(s):  
Rna Stability ◽  
Threshold Concentration ◽  
Ringspot Virus ◽  
Rna Transcripts ◽  
Cell Extracts ◽  
Confocal Immunofluorescence ◽  
Replicase Proteins ◽  
Double Label ◽  
Interfering Rna

The p36 and p95 proteins of Carnation Italian ringspot virus (CIRV), when expressed in Saccharomyces cerevisiae, supported the replication of defective interfering (DI) RNA. Double-label confocal immunofluorescence showed that both proteins localized to mitochondria, independently of each other. DI RNA progeny was localized by in situ hybridization both to mitochondria and to their proximity. Fractionation of cell extracts showed that replicase proteins associated with membranes with a consistent portion of DI RNA. DI RNA transcripts were stabilized more efficiently when co-expressed with both p36 and p95 than with either protein alone. By using the copper-inducible CUP1 promoter, p36 was shown to have an effect on DI RNA stability only above a threshold concentration, suggesting an ‘all-or-none’ behaviour. Conversely, the stabilizing activity of p95 was proportional to protein concentration in the range examined. Similarly, DI RNA replication level was proportional to p95 concentration and depended on a threshold concentration of p36.

scholarly journals De novo generation of cymbidium ringspot virus defective interfering RNA

1991 ◽  
Vol 72 (3) ◽  
pp. 505-509 ◽  
Author(s):  
J. Burgyan ◽  
L. Rubino ◽  
M. Russo
Keyword(s):  
De Novo ◽  
Ringspot Virus ◽  
Defective Interfering Rna ◽  
Interfering Rna

The yeast EUG1 gene encodes an endoplasmic reticulum protein that is functionally related to protein disulfide isomerase

1992 ◽  
Vol 12 (10) ◽  
pp. 4601-4611
Author(s):  
C Tachibana ◽  
T H Stevens
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Protein Disulfide Isomerase ◽  
Yeast Cells ◽  
Disulfide Isomerase ◽  
Protein Levels ◽  
Growth Defects ◽  
Endoplasmic Reticulum Protein ◽  
Related Proteins ◽  
Protein Disulfide

The product of the EUG1 gene of Saccharomyces cerevisiae is a soluble endoplasmic reticulum protein with homology to both the mammalian protein disulfide isomerase (PDI) and the yeast PDI homolog encoded by the essential PDI1 gene. Deletion or overexpression of EUG1 causes no growth defects under a variety of conditions. EUG1 mRNA and protein levels are dramatically increased in response to the accumulation of native or unglycosylated proteins in the endoplasmic reticulum. Overexpression of the EUG1 gene allows yeast cells to grow in the absence of the PDI1 gene product. Depletion of the PDI1 protein in Saccharomyces cerevisiae causes a soluble vacuolar glycoprotein to accumulate in its endoplasmic reticulum form, and this phenotype is only partially relieved by the overexpression of EUG1. Taken together, our results indicate that PDI1 and EUG1 encode functionally related proteins that are likely to be involved in interacting with nascent polypeptides in the yeast endoplasmic reticulum.

scholarly journals Altered intracellular calcium homeostasis and endoplasmic reticulum redox state in Saccharomyces cerevisiae cells lacking Grx6 glutaredoxin

2015 ◽  
Vol 26 (1) ◽  
pp. 104-116 ◽  
Author(s):  
Judit Puigpinós ◽  
Celia Casas ◽  
Enrique Herrero
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Intracellular Calcium ◽  
Calcium Homeostasis ◽  
Redox Regulation ◽  
Yeast Cells ◽  
Redox Equilibrium ◽  
Unfolded Protein ◽  
Reduced And Oxidized Glutathione ◽  
Thiol Oxidoreductase

Glutaredoxin 6 (Grx6) of Saccharomyces cerevisiae is an integral thiol oxidoreductase protein of the endoplasmic reticulum/Golgi vesicles. Its absence alters the redox equilibrium of the reticulum lumen toward a more oxidized state, thus compensating the defects in protein folding/secretion and cell growth caused by low levels of the oxidase Ero1. In addition, null mutants in GRX6 display a more intense unfolded protein response than wild-type cells upon treatment with inducers of this pathway. These observations support a role of Grx6 in regulating the glutathionylation of thiols of endoplasmic reticulum/Golgi target proteins and consequently the equilibrium between reduced and oxidized glutathione in the lumen of these compartments. A specific function influenced by Grx6 activity is the homeostasis of intracellular calcium. Grx6-deficient mutants have reduced levels of calcium in the ER lumen, whereas accumulation occurs at the cytosol from extracellular sources. This results in permanent activation of the calcineurin-dependent pathway in these cells. Some but not all the phenotypes of the mutant are coincident with those of mutants deficient in intracellular calcium transporters, such as the Golgi Pmr1 protein. The results presented in this study provide evidence for redox regulation of calcium homeostasis in yeast cells.

Genetic and biochemical evaluation of eucaryotic membrane protein topology: multiple transmembrane domains of Saccharomyces cerevisiae 3-hydroxy-3-methylglutaryl coenzyme A reductase

1990 ◽  
Vol 10 (2) ◽  
pp. 672-680
Author(s):  
C Sengstag ◽  
C Stirling ◽  
R Schekman ◽  
J Rine
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Coenzyme A ◽  
Transmembrane Domains ◽  
Protein Domain ◽  
Yeast Cells ◽  
Endoplasmic Reticulum Membrane ◽  
Hmg Coa Reductase ◽  
Coa Reductase ◽  
Histidinol Dehydrogenase

Both 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase isozymes of the yeast Saccharomyces cerevisiae are predicted to contain seven membrane-spanning domains. Previous work had established the utility of the histidinol dehydrogenase protein domain, encoded by HIS4C, as a topologically sensitive monitor that can be used to distinguish between the lumen of the endoplasmic reticulum and the cytoplasm. This study directly tested the structural predictions for HMG-CoA reductase by fusing the HIS4C domain to specific sites in the HMG-CoA reductase isozymes. Yeast cells containing the HMG-CoA reductase-histidinol dehydrogenase fusion proteins grew on histidinol-containing medium if the HIS4C domain was present on the cytoplasmic side of the endoplasmic reticulum membrane but not if the HIS4C domain was targeted to the endoplasmic reticulum lumen. Systematic exchanges of transmembrane domains between the isozymes confirmed that both isozymes had equivalent membrane topologies. In general, deletion of an even number of putative transmembrane domains did not interfere with the topology of the protein, but deletion or duplication of an odd number of transmembrane domains inverted the orientation of the protein. The data confirmed the earlier proposed topology for yeast HMG-CoA reductase, demonstrated that the yeast enzymes are core glycosylated, and provided in vivo evidence that the properties of transmembrane domains were, in part, dependent upon their context within the protein.

scholarly journals Expression of tombusvirus open reading frames 1 and 2 is sufficient for the replication of defective interfering, but not satellite, RNA

2004 ◽  
Vol 85 (10) ◽  
pp. 3115-3122 ◽  
Author(s):  
Luisa Rubino ◽  
Vitantonio Pantaleo ◽  
Beatriz Navarro ◽  
Marcello Russo
Keyword(s):  
Plant Cells ◽  
Open Reading Frames ◽  
Ringspot Virus ◽  
Yeast Cells ◽  
Deletion Mutants ◽  
Replication Proteins ◽  
Satellite Rna ◽  
Replicase Proteins ◽  
Replication Strategy ◽  
Reading Frames

Yeast cells co-expressing the replication proteins p36 and p95 of Carnation Italian ringspot virus (CIRV) support the RNA-dependent replication of several defective interfering (DI) RNAs derived from either the genome of CIRV or the related Cymbidium ringspot virus (CymRSV), but not the replication of a satellite RNA (sat RNA) originally associated with CymRSV. DI, but not sat RNA, was replicated in yeast cells co-expressing both DI and sat RNA. Using transgenic Nicotiana benthamiana plants constitutively expressing CymRSV replicase proteins (p33 and p92), or transiently expressing either these proteins or CIRV p36 and p95, it was shown that expression of replicase proteins alone was also not sufficient for the replication of sat RNA in plant cells. However, it was also shown that replicating CIRV genomic RNA deletion mutants encoding only replicase proteins could sustain replication of sat RNA in plant cells. These results suggest that sat RNA has a replication strategy differing from that of genomic and DI RNAs, for it requires the presence of a cis-replicating genome acting as a trans-replication enhancer.

scholarly journals A Defective Interfering RNA Molecule in Cymbidium Ringspot Virus Infections

1989 ◽  
Vol 70 (1) ◽  
pp. 235-239 ◽  
Author(s):  
J. Burgyan ◽  
F. Grieco ◽  
M. Russo
Keyword(s):  
Ringspot Virus ◽  
Virus Infections ◽  
Defective Interfering Rna ◽  
Interfering Rna

scholarly journals Gp78 Cooperates with RMA1 in Endoplasmic Reticulum-associated Degradation of CFTRΔF508

2008 ◽  
Vol 19 (4) ◽  
pp. 1328-1336 ◽  
Author(s):  
Daisuke Morito ◽  
Kazuyoshi Hirao ◽  
Yukako Oda ◽  
Nobuko Hosokawa ◽  
Fuminori Tokunaga ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Ubiquitin Ligase ◽  
Yeast Cells ◽  
Glutathione S Transferase ◽  
Assembly Factor ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Membrane Spanning ◽  
Interfering Rna

Misfolded or improperly assembled proteins in the endoplasmic reticulum (ER) are exported into the cytosol and degraded via the ubiquitin–proteasome pathway, a process termed ER-associated degradation (ERAD). Saccharomyces cerevisiae Hrd1p/Der3p is an ER membrane-spanning ubiquitin ligase that participates in ERAD of the cystic fibrosis transmembrane conductance regulator (CFTR) when CFTR is exogenously expressed in yeast cells. Two mammalian orthologues of yeast Hrd1p/Der3p, gp78 and HRD1, have been reported. Here, we demonstrate that gp78, but not HRD1, participates in ERAD of the CFTR mutant CFTRΔF508, by specifically promoting ubiquitylation of CFTRΔF508. Domain swapping experiments and deletion analysis revealed that gp78 binds to CFTRΔF508 through its ubiquitin binding region, the so-called coupling of ubiquitin to ER degradation (CUE) domain. Gp78 polyubiquitylated in vitro an N-terminal ubiquitin-glutathione-S-transferase (GST)-fusion protein, but not GST alone. This suggests that gp78 recognizes the ubiquitin that is already conjugated to CFTRΔF508 and catalyzes further polyubiquitylation of CFTRΔF508 in a manner similar to that of a multiubiquitin chain assembly factor (E4). Furthermore, we revealed by small interfering RNA methods that the ubiquitin ligase RMA1 functioned as an E3 enzyme upstream of gp78. Our data demonstrates that gp78 cooperates with RMA1 with E4-like activity in the ERAD of CFTRΔF508.

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