scholarly journals P-Body Components Are Required for Ty1 Retrotransposition during Assembly of Retrotransposition-Competent Virus-Like Particles

2009 ◽  
Vol 30 (2) ◽  
pp. 382-398 ◽  
Author(s):  
Mary Ann Checkley ◽  
Kunio Nagashima ◽  
Stephen J. Lockett ◽  
Katherine M. Nyswaner ◽  
David J. Garfinkel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Immunofluorescence Microscopy ◽  
Wild Type ◽  
Virus Like Particles ◽  
P Bodies ◽  
Cellular Processes ◽  
Body Component ◽  
Body Components ◽  
Ty1 Retrotransposition

ABSTRACT Ty1 is a retrovirus-like retrotransposon whose replication is influenced by diverse cellular processes in Saccharomyces cerevisiae. We have identified cytoplasmic P-body components encoded by DHH1, KEM1, LSM1, and PAT1 as cofactors that posttranscriptionally enhance Ty1 retrotransposition. Using fluorescent in situ hybridization and immunofluorescence microscopy, we found that Ty1 mRNA and Gag colocalize to discrete cytoplasmic foci in wild-type cells. These foci, which are distinct from P-bodies, do not form in P-body component mutants or under conditions suboptimal for retrotransposition. Our immunoelectron microscopy (IEM) data suggest that mRNA/Gag foci are sites where virus-like particles (VLPs) cluster. Overexpression of Ty1 leads to a large increase in retrotransposition in wild-type cells, which allows VLPs to be detected by IEM. However, retrotransposition is still reduced in P-body component mutants under these conditions. Moreover, the percentage of Ty1 mRNA/Gag foci and VLP clusters and levels of integrase and reverse transcriptase are reduced in these mutants. Ty1 antisense RNAs, which have been reported to inhibit Ty1 transposition, are more abundant in the kem1Δ mutant and colocalize with Ty1 mRNA in the cytoplasm. Therefore, Kem1p may prevent the aggregation of Ty1 antisense and mRNAs. Overall, our results suggest that P-body components enhance the formation of retrotransposition-competent Ty1 VLPs.

scholarly journals Thermolabile L-A virus-like particles from pet18 mutants of Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (2) ◽  
pp. 404-410 ◽  
Author(s):  
T Fujimura ◽  
R B Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Polymerase Activity ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Rna Dependent Rna Polymerase ◽  
Virus Like Particles ◽  
Rna Polymerase Activity ◽  
Lines Of Evidence

pet18 mutations in Saccharomyces cerevisiae confer on the cell the inability to maintain either L-A or M double-stranded RNAs (dsRNAs) at the nonpermissive temperature. In in vitro experiments, we examined the effects of pet18 mutations on the RNA-dependent RNA polymerase activity associated with virus-like particles (VLPs). pet18 mutations caused thermolabile RNA polymerase activity of L-A VLPs, and this thermolability was found to be due to the instability of the L-A VLP structure. The pet18 mutations did not affect RNA polymerase activity of M VLPs. Furthermore, the temperature sensitivity of wild-type L-A RNA polymerase differed substantially from that of M RNA polymerase. From these results, and from other genetic and biochemical lines of evidence which suggest that replication of M dsRNA requires the presence of L-A dsRNA, we propose that the primary effect of the pet18 mutation is on the L-A VLP structure and that the inability of pet18 mutants to maintain M dsRNA comes from the loss of L-A dsRNA.

scholarly journals Improving 3-methylphenol (m-cresol) production in yeast via in vivo glycosylation or methylation

2020 ◽  
Vol 20 (8) ◽  
Author(s):  
Julia Hitschler ◽  
Eckhard Boles
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Wild Type ◽  
Biotechnological Production ◽  
Yeast Saccharomyces Cerevisiae ◽  
In Situ Extraction ◽  
Methylsalicylic Acid Synthase ◽  
In The Wild

ABSTRACT Heterologous expression of 6-methylsalicylic acid synthase (MSAS) together with 6-MSA decarboxylase enables de novo production of the platform chemical and antiseptic additive 3-methylphenol (3-MP) in the yeast Saccharomyces cerevisiae. However, toxicity of 3-MP prevents higher production levels. In this study, we evaluated in vivo detoxification strategies to overcome limitations of 3-MP production. An orcinol-O-methyltransferase from Chinese rose hybrids (OOMT2) was expressed in the 3-MP producing yeast strain to convert 3-MP to 3-methylanisole (3-MA). Together with in situ extraction by dodecane of the highly volatile 3-MA this resulted in up to 211 mg/L 3-MA (1.7 mM) accumulation. Expression of a UDP-glycosyltransferase (UGT72B27) from Vitis vinifera led to the synthesis of up to 533 mg/L 3-MP as glucoside (4.9 mM). Conversion of 3-MP to 3-MA and 3-MP glucoside was not complete. Finally, deletion of phosphoglucose isomerase PGI1 together with methylation or glycosylation and feeding a fructose/glucose mixture to redirect carbon fluxes resulted in strongly increased product titers, with up to 897 mg/L 3-MA/3-MP (9 mM) and 873 mg/L 3-MP/3-MP as glucoside (8.1 mM) compared to less than 313 mg/L (2.9 mM) product titers in the wild type controls. The results show that methylation or glycosylation are promising tools to overcome limitations in further enhancing the biotechnological production of 3-MP.

Differential proteome–metabolome profiling of YCA1-knock-out and wild type cells reveals novel metabolic pathways and cellular processes dependent on the yeast metacaspase

2015 ◽  
Vol 11 (6) ◽  
pp. 1573-1583 ◽  
Author(s):  
Maša Ždralević ◽  
Valentina Longo ◽  
Nicoletta Guaragnella ◽  
Sergio Giannattasio ◽  
Anna Maria Timperio ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Stress Response ◽  
Metabolic Pathways ◽  
Cell Metabolism ◽  
Wild Type ◽  
Knock Out ◽  
Cellular Processes ◽  
Metabolome Profiling ◽  
Metabolomic Approach

A combined proteomic and metabolomic approach revealed new non-apoptotic roles of the metacaspaseYCA1gene inSaccharomyces cerevisiae, highlighting its involvement in the cell metabolism and stress response.

The CDC20 gene product of Saccharomyces cerevisiae, a beta-transducin homolog, is required for a subset of microtubule-dependent cellular processes

1991 ◽  
Vol 11 (11) ◽  
pp. 5592-5602
Author(s):  
N Sethi ◽  
M C Monteagudo ◽  
D Koshland ◽  
E Hogan ◽  
D J Burke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Product ◽  
Immunoblot Analysis ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cellular Processes ◽  
Chromosome Separation ◽  
G1 Cells ◽  
Dependent Processes

Previous analysis of cdc20 mutants of the yeast Saccharomyces cerevisiae suggests that the CDC20 gene product (Cdc20p) is required for two microtubule-dependent processes, nuclear movements prior to anaphase and chromosome separation. Here we report that cdc20 mutants are defective for a third microtubule-mediated event, nuclear fusion during mating of G1 cells, but appear normal for a fourth microtubule-dependent process, nuclear migration after DNA replication. Therefore, Cdc20p is required for a subset of microtubule-dependent processes and functions at multiple stages in the life cycle. Consistent with this interpretation, we find that cdc20 cells arrested by alpha-factor or at the restrictive temperature accumulate anomalous microtubule structures, as detected by indirect immunofluorescence. The anomalous microtubule staining patterns are due to cdc20 because intragenic revertants that revert the temperature sensitivity have normal microtubule morphologies. cdc20 mutants have a sevenfold increase in the intensity of antitubulin fluorescence in intranuclear spindles compared with spindles from wild-type cells, yet the total amount of tubulin is indistinguishable by Western immunoblot analysis. This result suggests that Cdc20p modulates microtubule structure in wild-type cells either by promoting microtubule disassembly or by altering the surface of the microtubules. Finally, we cloned and sequenced CDC20 and show that it encodes a member of a family of proteins that share homology to the beta subunit of transducin.

Recycling of the yeast v-SNARE Sec22p involves COPI-proteins and the ER transmembrane proteins Ufe1p and Sec20p

1998 ◽  
Vol 111 (11) ◽  
pp. 1507-1520 ◽  
Author(s):  
W. Ballensiefen ◽  
D. Ossipov ◽  
H.D. Schmitt
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Immunofluorescence Microscopy ◽  
Transmembrane Proteins ◽  
Retrograde Transport ◽  
Subcellular Fractionation ◽  
Hybrid Protein ◽  
Wild Type ◽  
Alpha Factor ◽  
Membrane Compartments

Vesicle-specific SNAP receptors (v-SNAREs) are believed to cycle between consecutive membrane compartments. The v-SNARE Sec22(Sly2)p mediates the targeting of vesicles between endoplasmic reticulum (ER) and early Golgi of Saccharomyces cerevisiae. To analyze factors involved in targeting of Sec22(Sly2)p, an alpha-factor-tagged Sec22 protein (Sec22-alpha) was employed. Only on reaching the late Golgi, can alpha-factor be cleaved from this hybrid protein by Kex2p, a protease localized in this compartment. In wild-type cells Kex2p-cleavage is observed only when Sec22-alpha is greatly overproduced. Immunofluorescence microscopy and subcellular fractionation studies showed that Sec22-alpha is returned to the ER from the late Golgi (Kex2p) compartment. When Sec22-alpha is expressed in wild-type cells at levels comparable to the quantities of endogenous Sec22p, very little of this protein is cleaved by Kex2p. Efficient cleavage, however, occurs in mutants defective in the retrograde transport of different ER-resident proteins indicating that Sec22-alpha rapidly reaches the late Golgi of these cells. These mutants (sec20-1, sec21-1, sec27-1 and ufe1-1) reveal Golgi structures when stained for Sec22-alpha and do not show the ER-immunofluorescence observed in wild-type cells. These results show consistently that Sec22p recycles from the Golgi back to the ER and that this recycling involves retrograde COPI vesicles.

scholarly journals A rare tRNA-Arg(CCU) that regulates Ty1 element ribosomal frameshifting is essential for Ty1 retrotransposition in Saccharomyces cerevisiae.

Genetics ◽  
1993 ◽  
Vol 135 (2) ◽  
pp. 309-320 ◽  
Author(s):  
K Kawakami ◽  
S Pande ◽  
B Faiola ◽  
D P Moore ◽  
J D Boeke ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Processing ◽  
Ribonuclease H ◽  
Wild Type ◽  
Ribosomal Frameshifting ◽  
Translational Frameshifting ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Ty1 Retrotransposition ◽  
Rate Limiting

Abstract Translation of the yeast retrotransposon Ty1 TYA1(gag)-TYB1(pol) gene occurs by a +1 ribosomal frameshifting event at the sequence CUU AGG C. Because overexpression of a low abundance tRNA-Arg(CCU) encoded by the HSX1 gene resulted in a reduction in Ty1 frameshifting, it was suggested that a translational pause at the AGG-Arg codon is required for optimum frameshifting. The present work shows that the absence of tRNA-Arg(CCU) affects Ty1 transposition, translational frameshifting, and accumulation of mature TYB1 proteins. Transposition of genetically tagged Ty1 elements decreases at least 50-fold and translational frameshifting increases 3-17-fold in cells lacking tRNA-Arg(CCU). Accumulation of Ty1-integrase and Ty1-reverse transcriptase/ribonuclease H is defective in an hsx1 mutant. The defect in Ty1 transposition is complemented by the wild-type HSX1 gene or a mutant tRNA-Arg(UCU) gene containing a C for T substitution in the first position of the anticodon. Overexpression of TYA1 stimulates Ty1 transposition 50-fold above wild-type levels when the level of transposition is compared in isogenic hsx1 and HSX1 strains. Thus, the HSX1 gene determines the ratio of the TYA1 to TYA1-TYB1 precursors required for protein processing or stability, and keeps expression of TYB1 a rate-limiting step in the retrotransposition cycle.

scholarly journals SPT5, an essential gene important for normal transcription in Saccharomyces cerevisiae, encodes an acidic nuclear protein with a carboxy-terminal repeat.

1991 ◽  
Vol 11 (6) ◽  
pp. 3009-3019 ◽  
Author(s):  
M S Swanson ◽  
E A Malone ◽  
F Winston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Protein ◽  
Immunofluorescence Microscopy ◽  
Essential Gene ◽  
Wild Type ◽  
Indirect Immunofluorescence Microscopy ◽  
Number Of Genes ◽  
Carboxy Terminal ◽  
Beta Galactosidase ◽  
Insertion Mutations

Mutations in the SPT5 gene of Saccharomyces cerevisiae were isolated previously as suppressors of delta insertion mutations at HIS4 and LYS2. In this study we have shown that spt5 mutations suppress the his4-912 delta and lys2-128 delta alleles by altering transcription. We cloned the SPT5 gene and found that either an increase or a decrease in the copy number of the wild-type SPT5 gene caused an Spt- phenotype. Construction and analysis of an spt5 null mutation demonstrated that SPT5 is essential for growth, suggesting that SPT5 may be required for normal transcription of a large number of genes. The SPT5 DNA sequence was determined; it predicted a 116-kDa protein with an extremely acidic amino terminus and a novel six-amino-acid repeat at the carboxy terminus (consensus = S-T/A-W-G-G-A/Q). By indirect immunofluorescence microscopy we showed that a bifunctional SPT5-beta-galactosidase protein was located in the yeast nucleus. This molecular analysis of the SPT5 gene revealed a number of interesting similarities to the previously characterized SPT6 gene of S. cerevisiae. These results suggest that SPT5 and SPT6 act in a related fashion to influence essential transcriptional processes in S. cerevisiae.

scholarly journals A new kinesin-like protein (Klp1) localized to a single microtubule of the Chlamydomonas flagellum.

1994 ◽  
Vol 125 (6) ◽  
pp. 1313-1326 ◽  
Author(s):  
M Bernstein ◽  
P L Beech ◽  
S G Katz ◽  
J L Rosenbaum
Keyword(s):  
Immunogold Labeling ◽  
Wild Type ◽  
Tail Region ◽  
Central Pair ◽  
Whole Cells ◽  
Cellular Processes ◽  
The Core ◽  
Kinesin Superfamily ◽  
Whole Mounts

The kinesin superfamily of mechanochemical proteins has been implicated in a wide variety of cellular processes. We have begun studies of kinesins in the unicellular biflagellate alga, Chlamydomonas reinhardtii. A full-length cDNA, KLP1, has been cloned and sequenced, and found to encode a new member of the kinesin superfamily. An antibody was raised against the nonconserved tail region of the Klp1 protein, and it was used to probe for Klp1 in extracts of isolated flagella and in situ. Immunofluorescence of whole cells indicated that Klp1 was present in both the flagella and cell bodies. In wild-type flagella, Klp1 was found tightly to the axoneme; immunogold labeling of wild-type axonemal whole mounts showed that Klp1 was restricted to one of the two central pair microtubules at the core of the axoneme. Klp1 was absent from the flagella of mutants lacking the central pair microtubules, but was present in mutant flagella from pf16 cells, which contain an unstable C1 microtubule, indicating that Klp1 was bound to the C2 central pair microtubule. Localization of Klp1 to the C2 microtubule was confirmed by immunogold labeling of negatively stained and thin-sectioned axonemes. These findings suggest that Klp1 may play a role in rotation or twisting of the central pair microtubules.

scholarly journals The small GTP-binding protein Rho1p is localized on the Golgi apparatus and post-Golgi vesicles in Saccharomyces cerevisiae.

1991 ◽  
Vol 115 (2) ◽  
pp. 309-319 ◽  
Author(s):  
M McCaffrey ◽  
J S Johnson ◽  
B Goud ◽  
A M Myers ◽  
J Rossier ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Golgi Apparatus ◽  
Immunofluorescence Microscopy ◽  
Cell Periphery ◽  
Related Protein ◽  
Wild Type ◽  
Golgi Vesicles ◽  
Golgi Transport ◽  
Transport Vesicles ◽  
Punctate Pattern

In Saccharomyces cerevisiae the ras-related protein Rho1p is essentially the only target for ADP-ribosylation by exoenzyme C3 of Clostridium botulinum. Using C3 to detect Rho1p in subcellular fractions, Rho1p was found primarily in the 10,000 g pellet (P2) containing large organelles; small amounts also were detected in the 100,000 g pellet (P3), and cytosol. When P2 organelles were separated in sucrose density gradients Rho1p comigrated with the Kex-2 activity, a late Golgi marker. Rho1p distribution was shifted from P2 to P3 in several mutants that accumulate post-Golgi vesicles. Rho1p comigrated with post-Golgi transport vesicles during fractionation of P3 organelles from wild-type or sec6 cells. Vesicles containing Rho1p were of the same size but different density than those bearing Sec4p, a ras-related protein located both on post-Golgi vesicles and the plasma membrane. Immunofluorescence microscopy detected Rho1p as a punctate pattern, with signal concentrated towards the cell periphery and in the bud. Thus, in S. cerevisiae Rho1p resides primarily in the Golgi apparatus, and also in vesicles that are likely to be early post-Golgi vesicles.

scholarly journals Heterogeneous functional Ty1 elements are abundant in the Saccharomyces cerevisiae genome.

Genetics ◽  
1994 ◽  
Vol 136 (4) ◽  
pp. 1245-1259 ◽  
Author(s):  
M J Curcio ◽  
D J Garfinkel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rare Event ◽  
Yeast Genome ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Negative Effect ◽  
In Trans ◽  
Ty1 Retrotransposition ◽  
In Cis ◽  
Restriction Site Polymorphisms

Abstract Despite the abundance of Ty1 RNA in Saccharomyces cerevisiae, Ty1 retrotransposition is a rare event. To determine whether transpositional dormancy is the result of defective Ty1 elements, functional and defective alleles of the retrotransposon in the yeast genome were quantitated. Genomic Ty1 elements were isolated by gap repair-mediated recombination of pGTy1-H3(delta 475-3944) HIS3, a multicopy plasmid containing a GAL1/Ty1-H3 fusion element lacking most of the gag domain (TYA) and the protease (PR) and integrase (IN) domains. Of 39 independent gap repaired pGTyHIS3 elements isolated, 29 (74%) transposed at high levels following galactose induction. The presence of restriction site polymorphisms within the gap repaired region of the 29 functional pGTyHIS3 elements indicated that they were derived from at least eight different genomic Ty1 elements and one Ty2 element. Of the 10 defective pGTyHIS3 elements, one was a partial gap repair event while the other nine were derived from at least six different genomic Ty1 elements. These results suggest that most genomic Ty1 elements encode functional TYA, PR and IN proteins. To understand how functional Ty1 elements are regulated, we tested the hypothesis that a TYB protein associates preferentially in cis with the RNA template that encodes it, thereby promoting transposition of its own element. A genomic Ty1 mhis3AI element containing either an in-frame insertion in PR or a deletion in TYB transposed at the same rate as a wild-type Ty1mhis3AI allele, indicating that TYB proteins act efficiently in trans. This result suggests in principle that defective genomic Ty1 elements could encode trans-acting repressors of transposition; however, expression of only one of the nine defective pGTy1 isolates had a negative effect on genomic Ty1 mhis3AI element transposition in trans, and this effect was modest. Therefore, the few defective Ty1 elements in the genome are not responsible for transpositional dormancy.

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