scholarly journals Adaptive and degenerative evolution of the S-Phase Kinase-Associated Protein 1-Like family in Arabidopsis thaliana

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6740 ◽  
Author(s):  
Zhihua Hua ◽  
Zhenyu Gao
Keyword(s):  
Sequence Variation ◽  
Biological Function ◽  
Gene Families ◽  
26S Proteasome ◽  
Yeast Two Hybrid ◽  
Transcriptional Suppression ◽  
Genomic Studies ◽  
Suppression Mechanism ◽  
Two Hybrid ◽  
Proteasome Protein

Genome sequencing has uncovered tremendous sequence variation within and between species. In plants, in addition to large variations in genome size, a great deal of sequence polymorphism is also evident in several large multi-gene families, including those involved in the ubiquitin-26S proteasome protein degradation system. However, the biological function of this sequence variation is yet not clear. In this work, we explicitly demonstrated a single origin of retroposed Arabidopsis Skp1-Like (ASK) genes using an improved phylogenetic analysis. Taking advantage of the 1,001 genomes project, we here provide several lines of polymorphism evidence showing both adaptive and degenerative evolutionary processes in ASK genes. Yeast two-hybrid quantitative interaction assays further suggested that recent neutral changes in the ASK2 coding sequence weakened its interactions with some F-box proteins. The trend that highly polymorphic upstream regions of ASK1 yield high levels of expression implied negative expression regulation of ASK1 by an as-yet-unknown transcriptional suppression mechanism, which may contribute to the polymorphic roles of Skp1-CUL1-F-box complexes. Taken together, this study provides new evolutionary evidence to guide future functional genomic studies of SCF-mediated protein ubiquitylation.

scholarly journals Regulation of Glycosylphosphatidylinositol-Anchored Protein (GPI-AP) Expression by F-Box/LRR-Repeat (FBXL) Protein in Wheat (Triticum aestivum L.)

Plants ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1606
Author(s):  
Min Jeong Hong ◽  
Jin-Baek Kim ◽  
Yong Weon Seo ◽  
Dae Yeon Kim
Keyword(s):  
Triticum Aestivum ◽  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Expression Patterns ◽  
Triticum Aestivum L ◽  
26S Proteasome ◽  
Bimolecular Fluorescence Complementation ◽  
Yeast Two Hybrid ◽  
Scf Complex ◽  
Two Hybrid

F-box proteins are substrate recognition components of the Skp1-Cullin-F-box (SCF) complex, which performs many important biological functions including the degradation of numerous proteins via the ubiquitin–26S proteasome system. In this study, we isolated the gene encoding the F-box/LRR-repeat (FBXL) protein from wheat (Triticum aestivum L.) seedlings and validated that the TaFBXL protein is a component of the SCF complex. Yeast two-hybrid assays revealed that TaFBXL interacts with the wheat glycosylphosphatidylinositol-anchored protein (TaGPI-AP). The green fluorescent protein (GFP) fusion protein of TaFBXL was detected in the nucleus and plasma membrane, whereas that of TaGPI-AP was observed in the cytosol and probably also plasma membrane. yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays revealed that TaFBXL specifically interacts with TaGPI-AP in the nucleus and plasma membrane, and TaGPI-AP is targeted by TaFBXL for degradation via the 26S proteasome system. In addition, TaFBXL and TaGPI-AP showed antagonistic expression patterns upon treatment with indole-3-acetic acid (IAA), and the level of TaGPI-AP was higher in tobacco leaves treated with both MG132 (proteasome inhibitor) and IAA than in leaves treated with either MG132 or IAA. Taken together, our data suggest that TaFBXL regulates the TaGPI-AP protein level in response to exogenous auxin application.

scholarly journals Role of the Carboxy Terminus of Escherichia coli FtsA in Self-Interaction and Cell Division

2000 ◽  
Vol 182 (22) ◽  
pp. 6366-6373 ◽  
Author(s):  
Lucía Yim ◽  
Guy Vandenbussche ◽  
Jesús Mingorance ◽  
Sonsoles Rueda ◽  
Mercedes Casanova ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Biological Function ◽  
Wild Type ◽  
Carboxy Terminus ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Carboxy Terminal ◽  
Two Hybrid

ABSTRACT The role of the carboxy terminus of the Escherichia coli cell division protein FtsA in bacterial division has been studied by making a series of short sequential deletions spanning from residue 394 to 420. Deletions as short as 5 residues destroy the biological function of the protein. Residue W415 is essential for the localization of the protein into septal rings. Overexpression of theftsA alleles harboring these deletions caused a coiled cell phenotype previously described for another carboxy-terminal mutation (Gayda et al., J. Bacteriol. 174:5362–5370, 1992), suggesting that an interaction of FtsA with itself might play a role in its function. The existence of such an interaction was demonstrated using the yeast two-hybrid system and a protein overlay assay. Even these short deletions are sufficient for impairing the interaction of the truncated FtsA forms with the wild-type protein in the yeast two-hybrid system. The existence of additional interactions between FtsA molecules, involving other domains, can be postulated from the interaction properties shown by the FtsA deletion mutant forms, because although unable to interact with the wild-type and with FtsAΔ1, they can interact with themselves and cross-interact with each other. The secondary structures of an extensive deletion, FtsAΔ27, and the wild-type protein are indistinguishable when analyzed by Fourier transform infrared spectroscopy, and moreover, FtsAΔ27 retains the ability to bind ATP. These results indicate that deletion of the carboxy-terminal 27 residues does not alter substantially the structure of the protein and suggest that the loss of biological function of the carboxy-terminal deletion mutants might be related to the modification of their interacting properties.

scholarly journals Genome-Wide Investigation of SBT Family Genes in Pineapple and Functional Analysis of AcoSBT1.12 in Floral Transition

2021 ◽  
Vol 12 ◽  
Author(s):  
Xingyue Jin ◽  
Yanhui Liu ◽  
Zhimin Hou ◽  
Yunfei Zhang ◽  
Yunying Fang ◽  
...  
Keyword(s):  
Biological Function ◽  
Proteolytic Enzymes ◽  
Floral Transition ◽  
Rna Seq ◽  
Yeast Two Hybrid ◽  
Element Analysis ◽  
Genome Wide ◽  
Delayed Flowering ◽  
Almost All ◽  
Two Hybrid

SBT (Subtilisin-like serine protease), a clan of serine proteolytic enzymes, plays a versatile role in plant growth and defense. Although SBT family genes have been obtained from studies of dicots such as Arabidopsis, little is known about the potential functions of SBT in the monocots. In this study, 54 pineapple SBT genes (AcoSBTs) were divided into six subfamilies and then identified to be experienced strong purifying selective pressure and distributed on 25 chromosomes unevenly. Cis-acting element analysis indicated that almost all AcoSBTs promoters contain light-responsive elements. Further, the expression pattern via RNA-seq data showed that different AcoSBTs were preferentially expressed in different above-ground tissues. Transient expression in tobacco showed that AcoSBT1.12 was located in the plasma membrane. Moreover, Transgenic Arabidopsis ectopically overexpressing AcoSBT1.12 exhibited delayed flowering time. In addition, under the guidance of bioinformatic prediction, we found that AcoSBT1.12 could interact with AcoCWF19L, AcoPUF2, AcoCwfJL, Aco012905, and AcoSZF1 by yeast-two hybrid (Y2H). In summary, this study provided valuable information on pineapple SBT genes and illuminated the biological function of AcoSBT1.12 in floral transition.

Identifications of DNA Sequences Encoding Key Region of SCR Interacting with SRK Extracellular Domain by Using Yeast Two-Hybrid System

2013 ◽  
Vol 38 (9) ◽  
pp. 1583-1591
Author(s):  
Li-Yan XUE ◽  
Bing LUO ◽  
Li-Quan ZHU ◽  
Yong-Jun YANG ◽  
He-Cui ZHANG ◽  
...  
Keyword(s):  
Hybrid System ◽  
Dna Sequences ◽  
Extracellular Domain ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Two Hybrid

DETECTION OF MICROCYSTINS BASED ON YEAST TWO-HYBRID SYSTEM

2008 ◽  
Vol 32 (2) ◽  
pp. 201-206
Author(s):  
Xiao PAN
Keyword(s):  
Hybrid System ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Two Hybrid

scholarly journals Structural Glycoprotein E2 of Classical Swine Fever Virus Interacts with Host Protein Dynactin Subunit 6 (DCTN6) during the Virus Infectious Cycle

2019 ◽  
Vol 94 (1) ◽  
Author(s):  
M. V. Borca ◽  
E. A. Vuono ◽  
E. Ramirez-Medina ◽  
P. Azzinaro ◽  
K. A. Berggren ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Interaction ◽  
Hybrid Approach ◽  
Classical Swine Fever ◽  
Host Protein ◽  
Yeast Two Hybrid ◽  
Protein Protein Interaction ◽  
Viral Virulence ◽  
Glycoprotein E2 ◽  
Two Hybrid

ABSTRACT The E2 protein in classical swine fever (CSF) virus (CSFV) is the major virus structural glycoprotein and is an essential component of the viral particle. E2 has been shown to be involved in several functions, including virus adsorption, induction of protective immunity, and virulence in swine. Using the yeast two-hybrid system, we previously identified a swine host protein, dynactin subunit 6 (DCTN6) (a component of the cell dynactin complex), as a specific binding partner for E2. We confirmed the interaction between DCTN6 and E2 proteins in CSFV-infected swine cells by using two additional independent methodologies, i.e., coimmunoprecipitation and proximity ligation assays. E2 residues critical for mediating the protein-protein interaction with DCTN6 were mapped by a reverse yeast two-hybrid approach using a randomly mutated E2 library. A recombinant CSFV mutant, E2ΔDCTN6v, harboring specific substitutions in those critical residues was developed to assess the importance of the E2-DCTN6 protein-protein interaction for virus replication and virulence in swine. CSFV E2ΔDCTN6v showed reduced replication, compared with the parental virus, in an established swine cell line (SK6) and in primary swine macrophage cultures. Remarkably, animals infected with CSFV E2ΔDCTN6v remained clinically normal during the 21-day observation period, which suggests that the ability of CSFV E2 to bind host DCTN6 protein efficiently during infection may play a role in viral virulence. IMPORTANCE Structural glycoprotein E2 is an important component of CSFV due to its involvement in many virus activities, particularly virus-host interactions. Here, we present the description and characterization of the protein-protein interaction between E2 and the swine host protein DCTN6 during virus infection. The E2 amino acid residues mediating the interaction with DCTN6 were also identified. A recombinant CSFV harboring mutations disrupting the E2-DCTN6 interaction was created. The effect of disrupting the E2-DCTN6 protein-protein interaction was studied using reverse genetics. It was shown that the same amino acid substitutions that abrogated the E2-DCTN6 interaction in vitro constituted a critical factor in viral virulence in the natural host, domestic swine. This highlights the potential importance of the E2-DCTN6 protein-protein interaction in CSFV virulence and provides possible mechanisms of virus attenuation for the development of improved CSF vaccines.

scholarly journals Identification of the Novel Host Protein Interacting With the Structural Protein VP1 of Chinese Sacbrood Virus by Yeast Two-Hybrid Screening

2019 ◽  
Vol 10 ◽  
Author(s):  
Xiyan Zhang ◽  
Dongliang Fei ◽  
Li Sun ◽  
Ming Li ◽  
YueYu Ma ◽  
...  
Keyword(s):  
Host Protein ◽  
The Novel ◽  
Structural Protein ◽  
Yeast Two Hybrid ◽  
Sacbrood Virus ◽  
Novel Host ◽  
Two Hybrid ◽  
Hybrid Screening

Determination of estrogenic activity in landfill leachate by simplified yeast two-hybrid assay

2002 ◽  
Vol 4 (6) ◽  
pp. 1040-1046 ◽  
Author(s):  
Yasunori Kawagoshi ◽  
Yukiko Tsukagoshi ◽  
Isao Fukunaga
Keyword(s):  
Landfill Leachate ◽  
Estrogenic Activity ◽  
Yeast Two Hybrid ◽  
Two Hybrid

scholarly journals The Zn-finger of Saccharomyces cerevisiae Rad18 and its adjacent region mediate interaction with Rad5

2021 ◽  
Author(s):  
Orsolya Frittmann ◽  
Vamsi K Gali ◽  
Miklos Halmai ◽  
Robert Toth ◽  
Zsuzsanna Gyorfy ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Ubiquitin Ligase ◽  
Dna Lesions ◽  
Template Switching ◽  
Adjacent Region ◽  
Yeast Two Hybrid ◽  
Replication Complex ◽  
Two Hybrid

Abstract DNA damages that hinder the movement of the replication complex can ultimately lead to cell death. To avoid that, cells possess several DNA damage bypass mechanisms. The Rad18 ubiquitin ligase controls error-free and mutagenic pathways that help the replication complex to bypass DNA lesions by monoubiquitylating PCNA at stalled replication forks. In Saccharomyces cerevisiae, two of the Rad18 governed pathways are activated by monoubiquitylated PCNA and they involve translesion synthesis polymerases, whereas a third pathway needs subsequent polyubiquitylation of the same PCNA residue by another ubiquitin ligase the Rad5 protein, and it employs template switching. The goal of this study was to dissect the regulatory role of the multidomain Rad18 in DNA damage bypass using a structure-function based approach. Investigating deletion and point mutant RAD18 variants in yeast genetic and yeast two-hybrid assays we show that the Zn-finger of Rad18 mediates its interaction with Rad5, and the N-terminal adjacent region is also necessary for Rad5 binding. Moreover, results of the yeast two-hybrid and in vivo ubiquitylation experiments raise the possibility that direct interaction between Rad18 and Rad5 might not be necessary for the function of the Rad5 dependent pathway. The presented data also reveal that yeast Rad18 uses different domains to mediate its association with itself and with Rad5. Our results contribute to better understanding of the complex machinery of DNA damage bypass pathways.

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