Detection of Protein Posttranslational Modifications from Whole-Cell Extracts in Saccharomyces cerevisiae

2014 ◽  
pp. 249-255 ◽  
Author(s):  
Lindsay G. Ball ◽  
Wei Xiao
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Posttranslational Modifications ◽  
Whole Cell ◽  
Cell Extracts ◽  
Protein Posttranslational Modifications

scholarly journals Identification of Kluyveromyces lactisTelomerase: Discontinuous Synthesis along the 30-Nucleotide-Long Templating Domain

1998 ◽  
Vol 18 (9) ◽  
pp. 4961-4970 ◽  
Author(s):  
Tracy Boswell Fulton ◽  
Elizabeth H. Blackburn
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Kluyveromyces Lactis ◽  
Telomerase Activity ◽  
Reaction Products ◽  
Telomeric Dna ◽  
Whole Cell ◽  
Cell Extracts ◽  
Dna Template ◽  
Insight Into

ABSTRACT Telomeres in the budding yeast Kluyveromyces lactisconsist of perfectly repeated 25-bp units, unlike the imprecise repeats at Saccharomyces cerevisiae telomeres and the short (6- to 8-bp) telomeric repeats found in many other eukaryotes. Telomeric DNA is synthesized by the ribonucleoprotein telomerase, which uses a portion of its RNA moiety as a template. K. lactistelomerase RNA, encoded by the TER1 gene, is ∼1.3 kb long and contains a 30-nucleotide templating domain, the largest ever examined. To examine the mechanism of polymerization by this enzyme, we identified and analyzed telomerase activity from K. lactiswhole-cell extracts. In this study, we exploited the length of the template and the precision of copying by K. lactistelomerase to examine primer elongation within one round of repeat synthesis. Under all in vitro conditions tested, K. lactistelomerase catalyzed only one round of repeat synthesis and remained bound to reaction products. We demonstrate that K. lactistelomerase polymerizes along the template in a discontinuous manner and stalls at two specific regions in the template. Increasing the amount of primer DNA-template RNA complementarity results in stalling, suggesting that the RNA-DNA hybrid is not unpaired during elongation in vitro and that lengthy duplexes hinder polymerization through particular regions of the template. We suggest that these observations provide an insight into the mechanism of telomerase and its regulation.

scholarly journals The Saccharomyces cerevisiae DNA polymerase alpha catalytic subunit interacts with Cdc68/Spt16 and with Pob3, a protein similar to an HMG1-like protein.

1997 ◽  
Vol 17 (7) ◽  
pp. 4178-4190 ◽  
Author(s):  
J Wittmeyer ◽  
T Formosa
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Polymerase ◽  
Catalytic Subunit ◽  
Amino Acid Sequence Similarity ◽  
Permissive Temperature ◽  
Whole Cell ◽  
Dna Polymerase Alpha ◽  
Cell Extracts ◽  
Cell Biol

We have used DNA polymerase alpha affinity chromatography to identify factors involved in eukaryotic DNA replication in the yeast Saccharomyces cerevisiae. Two proteins that bound to the catalytic subunit of DNA polymerase alpha (Pol1 protein) are encoded by the essential genes CDC68/SPT16 and POB3. The binding of both proteins was enhanced when extracts lacking a previously characterized polymerase binding protein, Ctf4, were used. This finding suggests that Cdc68 and Pob3 may compete with Ctf4 for binding to Pol1. Pol1 and Pob3 were coimmunoprecipitated from whole-cell extracts with antiserum directed against Cdc68, and Pol1 was immunoprecipitated from whole-cell extracts with antiserum directed against the amino terminus of Pob3, suggesting that these proteins may form a complex in vivo. CDC68 also interacted genetically with POL1 and CTF4 mutations; the maximum permissive temperature of double mutants was lower than for any single mutant. Overexpression of Cdc68 in a pol1 mutant strain dramatically decreased cell viability, consistent with the formation or modulation of an essential complex by these proteins in vivo. A mutation in CDC68/SPT16 had previously been shown to cause pleiotropic effects on the regulation of transcription (J. A. Prendergrast et al., Genetics 124:81-90, 1990; E. A. Malone et al., Mol. Cell. Biol. 11:5710-5717, 1991; A. Rowley et al., Mol. Cell. Biol. 11:5718-5726, 1991), with a spectrum of phenotypes similar to those caused by mutations in the genes encoding histone proteins H2A and H2B (Malone et al., Mol. Cell. Biol. 11:5710-5717, 1991). We show that at the nonpermissive temperature, cdc68-1 mutants arrest as unbudded cells with a 1C DNA content, consistent with a possible role for Cdc68 in the prereplicative stage of the cell cycle. The cdc68-1 mutation caused elevated rates of chromosome fragment loss, a phenotype characteristic of genes whose native products are required for normal DNA metabolism. However, this mutation did not affect the rate of loss or recombination for two intact chromosomes, nor did it affect the retention of a low-copy-number plasmid. The previously uncharacterized Pob3 sequence has significant amino acid sequence similarity with an HMG1-like protein from vertebrates. Based on these results and because Cdc68 has been implicated as a regulator of chromatin structure, we postulate that polymerase alpha may interact with these proteins to gain access to its template or to origins of replication in vivo.

scholarly journals Requirement for Three Novel Protein Complexes in the Absence of the Sgs1 DNA Helicase in Saccharomyces cerevisiae

Genetics ◽  
2001 ◽  
Vol 157 (1) ◽  
pp. 103-118 ◽  
Author(s):  
Janet R Mullen ◽  
Vivek Kaliraman ◽  
Samer S Ibrahim ◽  
Steven J Brill
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genome Stability ◽  
Protein Complexes ◽  
Ring Finger ◽  
Dna Helicase ◽  
Open Reading Frames ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Dna Helicases ◽  
Cell Extracts

Abstract The Saccharomyces cerevisiae Sgs1 protein is a member of the RecQ family of DNA helicases and is required for genome stability, but not cell viability. To identify proteins that function in the absence of Sgs1, a synthetic-lethal screen was performed. We obtained mutations in six complementation groups that we refer to as SLX genes. Most of the SLX genes encode uncharacterized open reading frames that are conserved in other species. None of these genes is required for viability and all SLX null mutations are synthetically lethal with mutations in TOP3, encoding the SGS1-interacting DNA topoisomerase. Analysis of the null mutants identified a pair of genes in each of three phenotypic classes. Mutations in MMS4 (SLX2) and SLX3 generate identical phenotypes, including weak UV and strong MMS hypersensitivity, complete loss of sporulation, and synthetic growth defects with mutations in TOP1. Mms4 and Slx3 proteins coimmunoprecipitate from cell extracts, suggesting that they function in a complex. Mutations in SLX5 and SLX8 generate hydroxyurea sensitivity, reduced sporulation efficiency, and a slow-growth phenotype characterized by heterogeneous colony morphology. The Slx5 and Slx8 proteins contain RING finger domains and coimmunoprecipitate from cell extracts. The SLX1 and SLX4 genes are required for viability in the presence of an sgs1 temperature-sensitive allele at the restrictive temperature and Slx1 and Slx4 proteins are similarly associated in cell extracts. We propose that the MMS4/SLX3, SLX5/8, and SLX1/4 gene pairs encode heterodimeric complexes and speculate that these complexes are required to resolve recombination intermediates that arise in response to DNA damage, during meiosis, and in the absence of SGS1/TOP3.

scholarly journals Development and Characterization of Two Types of Surface Displayed Levansucrases for Levan Biosynthesis

Catalysts ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 757
Author(s):  
Huiyi Shang ◽  
Danni Yang ◽  
Dairong Qiao ◽  
Hui Xu ◽  
Yi Cao
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Large Scale ◽  
Surface Display ◽  
Yeast Surface Display ◽  
Fusion Partner ◽  
Whole Cell ◽  
Food Industries ◽  
Yeast Surface ◽  
The Stability

Levan has wide applications in chemical, cosmetic, pharmaceutical and food industries. The free levansucrase is usually used in the biosynthesis of levan, but the poor reusability and low stability of free levansucrase have limited its large-scale use. To address this problem, the surface-displayed levansucrase in Saccharomyces cerevisiae were generated and evaluated in this study. The levansucrase from Zymomonas mobilis was displayed on the cell surface of Saccharomyces cerevisiae EBY100 using a various yeast surface display platform. The N-terminal fusion partner is based on a-agglutinin, and the C-terminal one is Flo1p. The yield of levan produced by these two whole-cell biocatalysts reaches 26 g/L and 34 g/L in 24 h, respectively. Meanwhile, the stability of the surface-displayed levansucrases is significantly enhanced. After six reuses, these two biocatalysts retained over 50% and 60% of their initial activities, respectively. Furthermore, the molecular weight and polydispersity test of the products suggested that the whole-cell biocatalyst of levansucrase displayed by Flo1p has more potentials in the production of levan with low molecular weight which is critical in certain applications. In conclusion, our method not only enable the possibility to reuse the enzyme, but also improves the stability of the enzyme.

scholarly journals E3 ligase Nedd4l promotes antiviral innate immunity by catalyzing K29-linked cysteine ubiquitination of TRAF3

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Peng Gao ◽  
Xianwei Ma ◽  
Ming Yuan ◽  
Yulan Yi ◽  
Guoke Liu ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Type I Interferon ◽  
Zinc Fingers ◽  
E3 Ligase ◽  
Type I ◽  
E3 Ligases ◽  
Protein Posttranslational Modifications ◽  
Antiviral Innate Immunity

AbstractUbiquitination is one of the most prevalent protein posttranslational modifications. Here, we show that E3 ligase Nedd4l positively regulates antiviral immunity by catalyzing K29-linked cysteine ubiquitination of TRAF3. Deficiency of Nedd4l significantly impairs type I interferon and proinflammatory cytokine production induced by virus infection both in vitro and in vivo. Nedd4l deficiency inhibits virus-induced ubiquitination of TRAF3, the binding between TRAF3 and TBK1, and subsequent phosphorylation of TBK1 and IRF3. Nedd4l directly interacts with TRAF3 and catalyzes K29-linked ubiquitination of Cys56 and Cys124, two cysteines that constitute zinc fingers, resulting in enhanced association between TRAF3 and E3 ligases, cIAP1/2 and HECTD3, and also increased K48/K63-linked ubiquitination of TRAF3. Mutation of Cys56 and Cys124 diminishes Nedd4l-catalyzed K29-linked ubiquitination, but enhances association between TRAF3 and the E3 ligases, supporting Nedd4l promotes type I interferon production in response to virus by catalyzing ubiquitination of the cysteines in TRAF3.

Targeted proteomics of myofilament phosphorylation and other protein posttranslational modifications

2014 ◽  
Vol 8 (7-8) ◽  
pp. 543-553 ◽  
Author(s):  
Genaro A. Ramirez-Correa ◽  
Maria Isabel Martinez-Ferrando ◽  
Pingbo Zhang ◽  
Anne M. Murphy
Keyword(s):  
Posttranslational Modifications ◽  
Targeted Proteomics ◽  
Protein Posttranslational Modifications

Interaction of the Repressors Nrg1 and Nrg2 With the Snf1 Protein Kinase in Saccharomyces cerevisiae

Genetics ◽  
2001 ◽  
Vol 158 (2) ◽  
pp. 563-572 ◽  
Author(s):  
Valmik K Vyas ◽  
Sergei Kuchin ◽  
Marian Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Hybrid System ◽  
Fluorescent Protein ◽  
Zinc Finger Protein ◽  
Glucose Repression ◽  
Snf1 Kinase ◽  
Cell Extracts ◽  
Green Fluorescent ◽  
Two Hybrid

Abstract The Snf1 protein kinase is essential for the transcription of glucose-repressed genes in Saccharomyces cerevisiae. We identified Nrg2 as a protein that interacts with Snf1 in the two-hybrid system. Nrg2 is a C2H2 zinc-finger protein that is homologous to Nrg1, a repressor of the glucose- and Snf1-regulated STA1 (glucoamylase) gene. Snf1 also interacts with Nrg1 in the two-hybrid system and co-immunoprecipitates with both Nrg1 and Nrg2 from cell extracts. A LexA fusion to Nrg2 represses transcription from a promoter containing LexA binding sites, indicating that Nrg2 also functions as a repressor. An Nrg1 fusion to green fluorescent protein is localized to the nucleus, and this localization is not regulated by carbon source. Finally, we show that VP16 fusions to Nrg1 and Nrg2 allow low-level expression of SUC2 in glucose-grown cells, and we present evidence that Nrg1 and Nrg2 contribute to glucose repression of the DOG2 gene. These results suggest that Nrg1 and Nrg2 are direct or indirect targets of the Snf1 kinase and function in glucose repression of a subset of Snf1-regulated genes.

Secretion-defective mutations in the signal sequence for Saccharomyces cerevisiae invertase

1986 ◽  
Vol 6 (7) ◽  
pp. 2382-2391
Author(s):  
C A Kaiser ◽  
D Botstein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Secretory Pathway ◽  
Signal Sequence ◽  
Mutant Gene ◽  
Yeast Cells ◽  
Molecular Weight Characteristic ◽  
Cell Extracts ◽  
Substitution Mutations ◽  
Membrane Components

Nine mutations in the signal sequence region of the gene specifying the secreted Saccharomyces cerevisiae enzyme invertase were constructed in vitro. The consequences of these mutations were studied after returning the mutated genes to yeast cells. Short deletions and two extensive substitution mutations allowed normal expression and secretion of invertase. Other substitution mutations and longer deletions blocked the formation of extracellular invertase. Yeast cells carrying this second class of mutant gene expressed novel active internal forms of invertase that exhibited the following properties. The new internal proteins had the mobilities in denaturing gels expected of invertase polypeptides that had retained a defective signal sequence and were otherwise unmodified. The large increase in molecular weight characteristic of glycosylation was not seen. On nondenaturing gels the mutant enzymes were found as heterodimers with a normal form of invertase that is known to be cytoplasmic, showing that the mutant forms of the enzyme are assembled in the same compartment as the cytoplasmic enzyme. All of the mutant enzymes were soluble and not associated with the membrane components after fractionation of crude cell extracts on sucrose gradients. Therefore, these signal sequence mutations result in the production of active internal invertase that has lost the ability to enter the secretory pathway. This demonstrates that the signal sequence is required for the earliest steps in membrane translocation.

Cap-dependent and cap-independent translation by internal initiation of mRNAs in cell extracts prepared from Saccharomyces cerevisiae

1994 ◽  
Vol 14 (11) ◽  
pp. 7322-7330 ◽  
Author(s):  
N Iizuka ◽  
L Najita ◽  
A Franzusoff ◽  
P Sarnow
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yeast Cells ◽  
In Vitro Translation ◽  
Translation System ◽  
Translational Efficiency ◽  
Coding Region ◽  
Initiation Mechanism ◽  
Noncoding Regions ◽  
Cell Extracts ◽  
Independent Translation

Translation extracts were prepared from various strains of Saccharomyces cerevisiae. The translation of mRNA molecules in these extracts were cooperatively enhanced by the presence of 5'-terminal cap structures and 3'-terminal poly(A) sequences. These cooperative effects could not be observed in other translation systems such as those prepared from rabbit reticulocytes, wheat germ, and human HeLa cells. Because the yeast translation system mimicked the effects of the cap structure and poly(A) tail on translational efficiency seen in vivo, this system was used to study cap-dependent and cap-independent translation of viral and cellular mRNA molecules. Both the 5' noncoding regions of hepatitis C virus and those of coxsackievirus B1 conferred cap-independent translation to a reporter coding region during translation in the yeast extracts; thus, the yeast translational apparatus is capable of initiating cap-independent translation. Although the translation of most yeast mRNAs was cap dependent, the unusually long 5' noncoding regions of mRNAs encoding cellular transcription factors TFIID and HAP4 were shown to mediate cap-independent translation in these extracts. Furthermore, both TFIID and HAP4 5' noncoding regions mediated translation of a second cistron when placed into the intercistronic spacer region of a dicistronic mRNA, indicating that these leader sequences can initiate translation by an internal ribosome binding mechanism in this in vitro translation system. This finding raises the possibility that an internal translation initiation mechanism exists in yeast cells for regulated translation of endogenous mRNAs.

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