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 Key role of the motor protein Kinesin 13B in the activity of homeodomain-leucine zipper I transcription factors

2020 ◽  
Vol 71 (20) ◽  
pp. 6282-6296
Author(s):  
Virginia Natali Miguel ◽  
Karina Fabiana Ribichich ◽  
Jorge Ignacio Giacomelli ◽  
Raquel Lia Chan
Keyword(s):  
Transcription Factors ◽  
Leucine Zipper ◽  
Molecular Mechanisms ◽  
Vascular Tissue ◽  
Expression Patterns ◽  
Motor Protein ◽  
Bimolecular Fluorescence Complementation ◽  
Yeast Two Hybrid ◽  
Cauline Leaf ◽  
Two Hybrid

Abstract The sunflower (Helianthus annuus) homeodomain-leucine zipper I transcription factor HaHB11 conferred differential phenotypic features when it was expressed in Arabidopsis, alfalfa, and maize plants. Such differences were increased biomass, seed yield, and tolerance to flooding. To elucidate the molecular mechanisms leading to such traits and identify HaHB11-interacting proteins, a yeast two-hybrid screening of an Arabidopsis cDNA library was carried out using HaHB11 as bait. The sole protein identified with high confidence as interacting with HaHB11 was Kinesin 13B. The interaction was confirmed by bimolecular fluorescence complementation and by yeast two-hybrid assay. Kinesin 13B also interacted with AtHB7, the Arabidopsis closest ortholog of HaHB11. Histochemical analyses revealed an overlap between the expression patterns of the three genes in hypocotyls, apical meristems, young leaves, vascular tissue, axillary buds, cauline leaves, and cauline leaf nodes at different developmental stages. AtKinesin 13B mutants did not exhibit a differential phenotype when compared with controls; however, both HaHB11 and AtHB7 overexpressor plants lost, partially or totally, their differential phenotypic characteristics when crossed with such mutants. Altogether, the results indicated that Kinesin 13B is essential for the homeodomain-leucine zipper transcription factors I to exert their functions, probably via regulation of the intracellular distribution of these transcription factors by the motor protein.

scholarly journals The Nuclear Migration Protein NUDF/LIS1 Forms a Complex with NUDC and BNFA at Spindle Pole Bodies

2008 ◽  
Vol 7 (6) ◽  
pp. 1041-1052 ◽  
Author(s):  
Kerstin Helmstaedt ◽  
Karen Laubinger ◽  
Katja Voßkuhl ◽  
Özgür Bayram ◽  
Silke Busch ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Fluorescent Protein ◽  
Affinity Purification ◽  
Nuclear Migration ◽  
Spindle Pole ◽  
Bimolecular Fluorescence Complementation ◽  
Yeast Two Hybrid ◽  
Spindle Pole Bodies ◽  
Green Fluorescent ◽  
Two Hybrid

ABSTRACTNuclear migration depends on microtubules, the dynein motor complex, and regulatory components like LIS1 and NUDC. We sought to identify new binding partners of the fungal LIS1 homolog NUDF to clarify its function in dynein regulation. We therefore analyzed the association between NUDF and NUDC inAspergillus nidulans. NUDF and NUDC directly interacted in yeast two-hybrid experiments via NUDF's WD40 domain. NUDC-green fluorescent protein (NUDC-GFP) was localized to immobile dots in the cytoplasm and at the hyphal cortex, some of which were spindle pole bodies (SPBs). We showed by bimolecular fluorescence complementation microscopy that NUDC directly interacted with NUDF at SPBs at different stages of the cell cycle. Applying tandem affinity purification, we isolated the NUDF-associated protein BNFA (forbinding toNUDF). BNFA was dispensable for growth and for nuclear migration. GFP-BNFA fusions localized to SPBs at different stages of the cell cycle. This localization depended on NUDF, since the loss of NUDF resulted in the cytoplasmic accumulation of BNFA. BNFA did not bind to NUDC in a yeast two-hybrid assay. These results show that the conserved NUDF and NUDC proteins play a concerted role at SPBs at different stages of the cell cycle and that NUDF recruits additional proteins specifically to the dynein complex at SPBs.

scholarly journals Hormonal Regulatory Patterns of LaKNOXs and LaBEL1 Transcription Factors Reveal Their Potential Role in Stem Bulblet Formation in LA Hybrid Lily

2021 ◽  
Vol 22 (24) ◽  
pp. 13502
Author(s):  
Yue Zhang ◽  
Zhen Zeng ◽  
Yubing Yong ◽  
Yingmin Lyu
Keyword(s):  
Potential Role ◽  
Transgenic Arabidopsis ◽  
Expression Patterns ◽  
Relative Increase ◽  
Bimolecular Fluorescence Complementation ◽  
Mechanism Of Formation ◽  
Yeast Two Hybrid ◽  
Physical Interactions ◽  
Two Hybrid ◽  
Strong Capacity

In lily reproduction, the mechanism of formation of bulbs has been a hot topic. However, studies on stem bulblet formation are limited. Stem bulblets, formed in the leaf axils of under- and above-ground stems, provide lilies with a strong capacity for self-propagation. First, we showed that above-ground stem bulblets can be induced by spraying 100 mg/L 6-BA on the LA hybrid lily ‘Aladdin’, with reduced endogenous IAA and GA4 and a higher relative content of cytokinins. Then, expression patterns of three potential genes (two KNOTTED1-like homeobox (KNOX) and one partial BEL1-like homeobox (BELL)), during stem bulblet formation from our previous study, were determined by RT-qPCR, presenting a down-up trend in KNOXs and a rising tendency in BELL. The partial BELL gene was cloned by RACE from L. ‘Aladdin’ and denoted LaBEL1. Physical interactions of LaKNOX1-LaBEL1 and LaKNOX1-LaKNOX2 were confirmed by yeast two-hybrid and bimolecular fluorescence complementation assays. Furthermore, hormonal regulatory patterns of single LaKNOX1, LaKNOX2, LaBEL1, and their heterodimers, were revealed in transgenic Arabidopsis, suggesting that the massive mRNA accumulations of LaKNOX1, LaKNOX2 and LaBEL1 genes during stem bulblet formation could cause the dramatic relative increase of cytokinins and the decline of GAs and IAA. Taken together, a putative model was proposed that LaKNOX1 interacts with LaKNOX2 and LaBEL1 to regulate multiple phytohormones simultaneously for an appropriate hormonal homeostasis, which suggests their potential role in stem bulblet formation in L. ‘Aladdin’.

scholarly journals Mammalian Homologue of the Caenorhabditis elegans UNC-76 Protein Involved in Axonal Outgrowth Is a Protein Kinase C ζ–interacting Protein

1999 ◽  
Vol 144 (3) ◽  
pp. 403-411 ◽  
Author(s):  
Shun'ichi Kuroda ◽  
Noritaka Nakagawa ◽  
Chiharu Tokunaga ◽  
Kenji Tatematsu ◽  
Katsuyuki Tanizawa
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Caenorhabditis Elegans ◽  
Protein Kinase ◽  
Rat Brain ◽  
Axonal Outgrowth ◽  
Yeast Two Hybrid ◽  
Interacting Protein ◽  
Kinase C ◽  
Two Hybrid

By the yeast two-hybrid screening of a rat brain cDNA library with the regulatory domain of protein kinase C ζ (PKCζ) as a bait, we have cloned a gene coding for a novel PKCζ-interacting protein homologous to the Caenorhabditis elegans UNC-76 protein involved in axonal outgrowth and fasciculation. The protein designated FEZ1 (fasciculation and elongation protein zeta-1) consisting of 393 amino acid residues shows a high Asp/Glu content and contains several regions predicted to form amphipathic helices. Northern blot analysis has revealed that FEZ1 mRNA is abundantly expressed in adult rat brain and throughout the developmental stages of mouse embryo. By the yeast two-hybrid assay with various deletion mutants of PKC, FEZ1 was shown to interact with the NH2-terminal variable region (V1) of PKCζ and weakly with that of PKCε. In the COS-7 cells coexpressing FEZ1 and PKCζ, FEZ1 was present mainly in the plasma membrane, associating with PKCζ and being phosphorylated. These results indicate that FEZ1 is a novel substrate of PKCζ. When the constitutively active mutant of PKCζ was used, FEZ1 was found in the cytoplasm of COS-7 cells. Upon treatment of the cells with a PKC inhibitor, staurosporin, FEZ1 was translocated from the cytoplasm to the plasma membrane, suggesting that the cytoplasmic translocation of FEZ1 is directly regulated by the PKCζ activity. Although expression of FEZ1 alone had no effect on PC12 cells, coexpression of FEZ1 and constitutively active PKCζ stimulated the neuronal differentiation of PC12 cells. Combined with the recent finding that a human FEZ1 protein is able to complement the function of UNC-76 necessary for normal axonal bundling and elongation within axon bundles in the nematode, these results suggest that FEZ1 plays a crucial role in the axon guidance machinery in mammals by interacting with PKCζ.

scholarly journals Inhibition of Ubiquitination and Stabilization of Human Ubiquitin E3 Ligase PIRH2 by Measles Virus Phosphoprotein

2005 ◽  
Vol 79 (18) ◽  
pp. 11824-11836 ◽  
Author(s):  
Mingzhou Chen ◽  
Jean-Claude Cortay ◽  
Ian R. Logan ◽  
Vasileia Sapountzi ◽  
Craig N. Robson ◽  
...  
Keyword(s):  
Binding Site ◽  
Measles Virus ◽  
Fluorescent Protein ◽  
E3 Ligase ◽  
Yeast Two Hybrid ◽  
Ubiquitin E3 Ligase ◽  
P Protein ◽  
Two Hybrid

ABSTRACT Using a C-terminal domain (PCT) of the measles virus (MV) phosphoprotein (P protein) as bait in a yeast two-hybrid screen, a cDNA identical to the recently described human p53-induced-RING-H2 (hPIRH2) cDNA was isolated. A glutathione S-transferase-hPIRH2 fusion protein expressed in bacteria was able to pull down P protein when mixed with an extract from P-expressing HeLa cells in vitro, and myc-tagged hPIRH2 could be reciprocally coimmunoprecipitated with MV P protein from human cells. Additionally, immunoprecipitation experiments demonstrated that hPIRH2-myc, MV P, and nucleocapsid (N) proteins form a ternary complex. The hPIRH2 binding site was mapped to the C-terminal X domain region of the P protein by using a yeast two-hybrid assay. The PCT binding site was mapped on hPIRH2 by using a novel yeast two-hybrid tagged PCR approach and by coimmunoprecipitation of hPIRH2 cysteine mutants and mouse/human PIRH2 chimeras. The hPIRH2 C terminus could mediate the interaction with MV P which was favored by the RING-H2 motif. When coexpressed with an enhanced green fluorescent protein-tagged hPIRH2 protein, MV P alone or in a complex with MV N was able to redistribute hPIRH2 to outside the nucleus, within intracellular aggregates. Finally, MV P efficiently stabilized hPIRH2-myc expression and prevented its ubiquitination in vivo but had no effect on the stability or ubiquitination of an alternative ubiquitin E3 ligase, Mdm2. Thus, MV P protein is the first protein from a pathogen that is able to specifically interact with and stabilize the ubiquitin E3 ligase hPIRH2 by preventing its ubiquitination.

scholarly journals The role of Clt1-regulated-Xylan metabolism in the melanin and toxin formation for the pathogenicity of Curvularia lunata in Maize

2021 ◽  
Author(s):  
Jinxin Gao ◽  
Jie Chen
Keyword(s):  
Toxin Production ◽  
Bimolecular Fluorescence Complementation ◽  
Curvularia Lunata ◽  
Yeast Two Hybrid ◽  
Acetyl Xylan Esterase ◽  
Btb Domain ◽  
Intermediate Metabolites ◽  
Regulative Mechanism ◽  
Two Hybrid

We previously reported that the BTB domain-containing protein Clt1 regulates melanin and toxin synthesis, conidiation, and pathogenicity in Curvularia lunata, but the interacting proteins and regulative mechanism of Clt1 are unclear. In this research, we identified two proteins, which respectively correspond to xylanase (Clxyn24) and acetyl xylan esterase (Claxe43) from C. lunata were regulated by Clt1. Yeast two-hybrid (Y2H), and bimolecular fluorescence complementation assays were conducted to verify the interaction of Clt1 with full-length Clxyn24 and Claxe43. Furthermore, the Y2H assay revealed that Clt1 physically interacted with Clxyn24 and Claxe43 through its BTB domain to degrade xylan which was used as a carbon source for C. lunata growth. The utilization of xylan provides acetyl-CoA for the synthesis of melanin and toxin, as well as energy and other intermediate metabolites for conidiation. Furthermore, transcriptome analysis revealed that PKS18 and its 13 flanking genes are found clustered in a region spanning 57.89 kb on scaffold 9 of the C. lunata CX-3 genome were down-regulated in toxin production deficient mutant T806, and this cluster is possibly responsible for toxin biosynthesis of C. lunata.

scholarly journals Expansion and Functional Diversification of SKP1-Like Genes in Wheat (Triticum aestivum L.)

2019 ◽  
Vol 20 (13) ◽  
pp. 3295
Author(s):  
Imen HajSalah El Beji ◽  
Said Mouzeyar ◽  
Mohammed-Fouad Bouzidi ◽  
Jane Roche
Keyword(s):  
Triticum Aestivum ◽  
Physcomitrella Patens ◽  
Expression Patterns ◽  
Protein S ◽  
Triticum Aestivum L ◽  
Wheat Genome ◽  
Genome Database ◽  
E3 Ligases ◽  
Expression Platform ◽  
Public Data

The ubiquitin proteasome 26S system (UPS), involving monomeric and multimeric E3 ligases is one of the most important signaling pathways in many organisms, including plants. The SCF (SKP1/Cullin/F-box) multimeric complex is particularly involved in response to development and stress signaling. The SKP1 protein (S-phase kinase-associated protein 1) is the core subunit of this complex. In this work, we firstly identified 92 and 87 non-redundant Triticum aestivum SKP1-like (TaSKP) genes that were retrieved from the latest release of the wheat genome database (International Wheat Genome Sequencing Consortium (IWGSC) RefSeq v1.0) and the genome annotation of the TGAC v1 respectively. We then investigated the structure, phylogeny, duplication events and expression patterns of the SKP1-like gene family in various tissues and environmental conditions using a wheat expression platform containing public data. TaSKP1-like genes were expressed differentially in response to stress conditions, displaying large genomic variations or short insertions/deletions which suggests functional specialization within TaSKP1-like genes. Finally, interactions between selected wheat FBX (F-box) proteins and putative ancestral TaSKP1-like proteins were tested using the yeast two-hybrid (Y2H) system to examine the molecular interactions. These observations suggested that six Ta-SKP1 genes are likely to be ancestral genes, having similar functions as ASK1 and ASK2 in Arabidopsis, OSK1 and OSK20 in rice and PpSKP1 and PpSKP2 in Physcomitrella patens.

CsSWEET1a and CsSWEET17 Mediate Growth and Freezing Tolerance by Promoting Sugar Transport across the Plasma Membrane

2020 ◽  
Vol 61 (9) ◽  
pp. 1669-1682
Author(s):  
Lina Yao ◽  
Changqing Ding ◽  
Xinyuan Hao ◽  
Jianming Zeng ◽  
Yajun Yang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Freezing Tolerance ◽  
Sugar Transport ◽  
Bimolecular Fluorescence Complementation ◽  
Freezing Stress ◽  
Sugar Uptake ◽  
Wild Type ◽  
Alternatively Spliced ◽  
Germination And Growth ◽  
Two Hybrid

Abstract Sugars Will Eventually be Exported Transporters (SWEETs) are important in plant biological processes. Expression levels of CsSWEET1a and CsSWEET17 are induced by cold acclimation (CA) and cold stress in Camellia sinensis. Here, we found that CsSWEET17 was alternatively spliced, and its exclusion (Ex) transcript was associated with the CA process. Both plasma membrane-localized CsSWEET1a and CsSWEET17 transport hexoses, but cytoplasm-localized CsSWEET17-Ex does not. These results indicate that alternative splicing may be involved in regulating the function of SWEET transporters in response to low temperature in plants. The extra C-terminal of CsSWEET17, which is not found in the tonoplast fructose transporter AtSWEET17, did not affect its plasma membrane localization but promoted its sugar transport activities. The overexpression (OE) of CsSWEET1a and CsSWEET17 genes resulted in an increased sugar uptake in Arabidopsis, affecting plant germination and growth. The leaf and seed sizes of the CsSWEET17-OE lines were significantly larger than those of the wild type. Moreover, the OE of CsSWEET1a and CsSWEET17 significantly reduced the relative electrolyte leakage levels under freezing stress. Compared with the wild type, the expression of AtCWINV genes was suppressed in both CsSWEET1a-OE and CsSWEET17-OE lines, indicating the alteration in sugar contents in the cell walls of the OE lines. Furthermore, the interaction between CsSWEET1a and CsSWEET17 was confirmed using yeast two-hybrid and bimolecular fluorescence complementation assays. We showed that CsSWEET1a and CsSWEET17 form homo-/heterodimers in the plasma membrane and mediate the partitioning of sugars between the cytoplasm and the apoplast, thereby regulating plant growth and freezing tolerance.

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